Chloroplast Expressing Colostrum or Milk Polypeptides

ABSTRACT

Provided are chloroplasts engineered to recombinantly express mammalian colostrum and milk polypeptides photosynthetic organisms containing such chloroplasts, and compositions comprising such organisms and methods for producing such organisms. In certain embodiments, provided is a chloroplast comprising one or more polynucleotides encoding one or more mammalian milk or colostrum polypeptides selected from osteopontin, lactadherin, cathelicidin-1, lysozyme, lactoperoxidase, lingual antimicrobial peptide (LAP), alpha-lactalbumin, and soluble CD14.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/135,559, filed on Apr. 22, 2016, and issued on Aug. 15, 2017 as U.S.Pat. No. 9,732,351, which a continuation under 35 U.S.C. §111 of Intl.Appl. No. PCT/US2015/016460, filed on Feb. 19, 2015, and claims thebenefit of and the right to priority to PCT/US2015/016460 under 35U.S.C. § 365. Intl. Appl. No. PCT/US2015/016460 claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Appl. No. 61/942,024, filed onFeb. 19, 2014, all of which are hereby incorporated herein by referencein their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 26, 2015, isnamed UCSDP033WO_SL.txt and is 91 kilobytes in size.

FIELD

Provided are colostrum and milk polypeptides recombinantly expressed inphotosynthetic organisms, compositions comprising such organisms andmethods for producing such organisms.

BACKGROUND

Colostrum or milk bioactive proteins have only been available from thenatural source (e.g., human and bovine colostrum or milk); becausenatural sources are in very limited supply, so too are the associatedbioactives contained in them. Tailored combinations of colostrum andmilk bioactives have never been available. Bioactive colostrum and milkproteins require both polypeptide accumulation and correct proteinfolding and post-translational modification.

SUMMARY

The invention provides a solution to drawbacks associated withconventional recombinant protein production methods. For example, themethods feature photosynthetic organisms such as the Chlamydomonasreinhartdii, a single-cell green alga, engineered to contain nucleicacids encoding a milk or colostrum protein in the chloroplasts. As aresult, the organism produces recombinant biologically active mammalianproteins in the chloroplast. Algae's ability to fold, assemble andaccumulate multiple domain proteins as soluble molecules withappropriate post-translational modification of phosphorylation, topreserve biological activity, offers significant advantages. Theorganisms, isolated cells, and/or sub-cellular organelles such aschloroplasts are useful to produce proteins, which are rare ornon-existent in the plant genome/proteome. The proteins produced can bedelivered without purification, compared to conventional bioreactorsystems, e.g., CHO, bacteria, or yeast, to yield bioactive compoundsuseful in an edible delivery system.

This edible delivery system comprises food as medicine for not onlyhuman therapy but also for veterinary use for companion animals such asdogs and cat as well as livestock such as cows, pigs, chickens, horsesand other performance as well as working animals. Purification ofbioactive proteins is expedited using the engineered organisms anddirect oral administration of organisms themselves leads to efficientdelivery, e.g., oral delivery, to gastrointestinal tissues. Oraldelivery leads to absorption and assimilation of the encoded proteinsinto tissues, e.g., bone tissue, of the subject to which the protein orrecombinant organism is administered. The system is particularly usefulto express/produce proteins, the biological activity of which depends onpost-translational modification such as phosphorylation.

Accordingly provided are photosynthetic organisms, e.g., algae andcyanobacteria, as well as cells purified from populations of suchorganisms and/or sub-cellular organelles, e.g., chloroplasts, purifiedor obtained from such organism. In varying embodiments, the chloroplastsor cyanobacteria comprise one or more polynucleotides encoding one ormore mammalian milk or colostrum polypeptides selected from osteopontin,lactadherin, cathelicidin-1, lysozyme, lactoperoxidase, lingualantimicrobial peptide (LAP), lactalbumin, and soluble CD14. In varyingembodiments, the chloroplast is from a photosynthetic organism. Invarying embodiments, the chloroplast is from a non-vascularphotosynthetic eukaryotic organism. In varying embodiments, thechloroplast is from a photosynthetic unicellular organism. In varyingembodiments, the chloroplast is from a microalgae. In some embodiments,the photosynthetic organism is selected from the group consisting ofChlorophyta (green algae), Rhodophyta (red algae), Stramenopiles(heterokonts), Xanthophyceae (yellow-green algae), Glaucocystophyceae(glaucocystophytes), Chlorarachniophyceae (chlorarachniophytes),Euglenida (euglenids), Haptophyceae (coccolithophorids), Chrysophyceae(golden algae), Cryptophyta (cryptomonads), Dinophyceae(dinoflagellates), Haptophyceae (coccolithophorids), Bacillariophyta(diatoms), Eustigmatophyceae (eustigmatophytes), Raphidophyceae(raphidophytes), Scenedesmaceae, Phaeophyceae (brown algae). In someembodiments, the photosynthetic organism is selected from the groupconsisting of Chlamydomonas reinhardtii, Dunaliella sauna, Haematococcuspluvialis, Chlorella vulgaris, Acutodesmus obliquus, Scenedesmusdimorphus, Arthrospira platensis, Arthrospira maxima, Anabaenasp.PCC7120, Leptolyngbya sp, Synechocystis sp, and Synechococcuselongates PCC7942. In some embodiments, the chloroplast is a Chlorophyta(green algae) chloroplast. In some embodiments, the green algae isselected from the group consisting of Chlamydomonas, Dunaliella,Haematococcus, Chlorella, and Scenedesmaceae. In some embodiments, theChlamydomonas is a Chlamydomonas reinhardtii. In varying embodiments,the green algae can be a Chlorophycean, a Chlamydomonas, C. reinhartdii,C. reinhartdii 137c, or a psbA deficient C. reinhartdii strain. Invarying embodiments, the chloroplast is from a higher plant selectedfrom Brassicaceae, Solanaceae, Phaseoleae, Zea and Oryzeae. In someembodiments, the chloroplast comprises at least two (e.g., at least 3,4, 5, 6, 7, 8, 9 or 10) polynucleotides encoding at least two mammalianmilk or colostrum polypeptides. In varying embodiments, the at least twomammalian milk or colostrum polypeptides comprise osteopontin andmammary associated serum amyloid A3 (MAA). In some embodiments, the oneor more mammalian polypeptides further comprises one or more mammalianmilk or colostrum polypeptides selected from mammary associated serumamyloid A3, osteopontin, soluble cluster of differentiation 14 (sCD14),lactedherin (milk fat globule-EGF factor 8 protein, Mfge8),alpha-lactalbumin, beta-lactoglobin, haptoglobin, immunoglobulins (e.g.,IgG1, IgG2, IgA, IgM, IgD), lactoferrin, proline rich polypeptide (PRP),proline rich polypeptide (PRP), growth factors (e.g., transforminggrowth factor (TGF)-β1, TGF-β2 insulin-like growth factor 1 (somatomedinC) (IGF-1), IGF-2, epidermal growth factor, heparin-binding epidermalgrowth factor-like growth factor, betacellulin), cytokines (e.g., IL-6,IL-1(3, IL-1ra), milk fat globule membrane (MFGM) proteins, serumalbumin, glycomacropeptide, casein proteins (e.g., β-casein, κ-casein,αs1-casein, αs2-casein and γ-casein), enzymes (e.g., superoxidedismutase, lactoperoxidase, alkaline phosphatase, platelet-activatingfactor-acetylhydroxylase, lysozyme, lipase), mucins, antimicrobialpeptides, alpha-defensins, beta-defensins, cathelicidins, 14-3-3 proteinzeta chain, 5-oxoprolinase (ATP-hydrolyzing), actin, cytoplasmic 1(beta-actin), adipose differentiation-related protein, albumin(precursor), aldehyde dehydrogenase (NAD) 2 precursor, ankyrin 3, nodeof Ranvier (ankyrin G), annexin 1, annexin A2, apolipoprotein A-I,apolipoprotein B, ARP3 (actin-related protein 3, yeast) homolog, ATPsynthase, H+ transporting, mitochondrial, F1 complex, alpha subunit,beta-2-microglobulin precursor (lactollin); butyrophilin, subfamily 1,member A1; capping protein (actin filament); muscle Z-line, alpha 1;casein kinase 1, alpha 1; coronin, actin binding protein, 1A; CD36antigen [collagen type I receptor, thrombospondin receptor];Chitinase-like protein 1 (CLP-1); DEAD (Asp-Glu-Ala-Asp) box polypeptide54; deleted in malignant brain tumors 1; diacylglycerol kinase kappa;endoplasmin precursor (GRP94/GP96); enolase 1; eukaryotic translationinitiation factor 4, gamma 2; fatty acid binding protein, heart-type(MDGI); fetuin; fibrinogen alpha chain; fibrinogen beta chain precursor;fibrinogen gamma-B chain precursor; gene model 440, (NCBI); glucoseregulated protein 58 kD; glutamate receptor, ionotropic, delta 1;glutathione S-transferase, mu 1; glyceraldehyde-3-phosphate;dehydrogenase (GAPDH); glycerol-3-phosphate dehydrogenase 2;glycoprotein antigen MGP57/53 (Lactadherin/bP47 protein);glycosylation-dependent cell adhesion molecule 1 (lactophorin/PP3);guanine nucleotide binding protein, beta 2; H3 histone, family 3A; heatshock 70 kDa protein 8; heat shock 70 kD protein 5 (glucose-regulatedprotein); heat shock protein 27; heat shock protein 70 kDa protein 1A;histone 2, H2ab; zinc finger protein 668; hypothetical/unnamed proteinLOC51063; IRTA2; isocitrate dehydrogenase 1 (NADP+), soluble; keratin 9;keratin complex 2, basic, gene 6a; keratin, type I cytoskeletal 10; andKIAA1586 protein. In varying embodiments, the one or more mammalianpolypeptides are bioactive. In some embodiments, the one or morepolynucleotides encoding the one or more mammalian polypeptides isintegrated into the chloroplast genome. In some embodiments, the one ormore mammalian polypeptides are human milk or colostrum polypeptides. Insome embodiments, the one or more mammalian polypeptides are milk orcolostrum polypeptides from a mammal selected from the group consistingof human, canine, feline, bovine, porcine, ovine and caprine. In someembodiments, the nucleic acid encoding osteopontin comprises apolynucleotide having at least about 60% sequence identity, e.g., atleast about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity, to SEQ ID NO:7. In some embodiments, the nucleic acid encodinglactadherin comprises a polynucleotide having at least about 60%sequence identity, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% sequence identity, to SEQ ID NO:9. In some embodiments,the nucleic acid encoding cathelicidin-1 comprises a polynucleotidehaving at least about 60% sequence identity, e.g., at least about 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, to SEQ IDNO:11. In some embodiments, the one or more mammalian polypeptides arephosphorylated. In varying embodiments, the one or more mammalianpolypeptides are bioactive and phosphorylated at 50% or more, e.g., 60%,70%, 80%, 90% or more, of the amino acid positions that arephosphorylated in the mammalian peptide expressed from a mammalian cell.In some embodiments, the one or more mammalian polypeptides comprisesbovine osteopontin and the bovine osteopontin is phosphorylated at oneor more amino acids comprising S45, S47, S218, S230, S241, S252 andS259, wherein the amino acid positions are with reference to SEQ ID NO:8and FIG. 14. In some embodiments, the bovine osteopontin is furtherphosphorylated at one or more amino acids comprising S48, T51, S85, S88,T93, T94, S100, S103, S106, S109 and S260, wherein the amino acidpositions are with reference to SEQ ID NO:8 and FIG. 14. In varyingembodiments, the one or more mammalian polypeptides comprises humanosteopontin and the human osteopontin is phosphorylated at one or moreamino acids comprising Ser20, Ser22, Ser23, Ser58, Ser60, Ser63, Ser81,Ser84, Ser90, Ser99, Ser102, Ser105, Ser108, Seri 11, Thr167, Ser173,Ser177, Ser197, Ser201, Ser206, Ser210, Ser216, Ser236, Ser245, Ser249,Ser252, Ser257, Ser273, Ser285, Ser290, and Ser292, wherein the aminoacid positions are with reference to FIGS. 3 and 4. In some embodiments,the one or more mammalian polypeptides comprises canine osteopontin andthe canine osteopontin is phosphorylated at one or more amino acidscomprising Thr57, Thr60, Ser153, Ser163, Thr164, Ser174, Ser176, Ser198,Ser207, Ser230, Ser233, Ser237, Ser246, Ser282, Ser289, and Ser290,wherein the amino acid positions are with reference to FIGS. 5 and 6. Insome embodiments, the one or more mammalian polypeptides comprisesfeline osteopontin and the feline osteopontin is phosphorylated at oneor more amino acids comprising Ser174, Ser176, Ser237, and Ser282,wherein the amino acid positions are with reference to FIGS. 7 and 8. Insome embodiments, the one or more polynucleotides are operably linked toa promoter that promotes expression in the chloroplast. In varyingembodiments, two or more polynucleotides encoding two or more mammalianmilk/colostrum polypeptides are integrated into the chloroplast genome.In varying embodiments, the one or more mammalian polypeptides areretained or sequestered in the chloroplast. In some embodiments, thechloroplast is intact. In some embodiments, the chloroplast isfreeze-dried. In varying embodiments, the one or more colostrum/milkpolypeptides are not purified or isolated from the chloroplast.

In a further aspect, provided are cells from a photosynthetic organism,the cells comprising one or more polynucleotides encoding one or moremammalian milk or colostrum polypeptides selected from osteopontin,lactadherin, soluble CD14, alpha-lactalbumin, lactoperoxidase, lysozyme,lingual antimicrobial peptide and cathelicidin-1. In varyingembodiments, the cell is from a non-vascular photosynthetic eukaryoticorganism. In varying embodiments, the cell is from a photosyntheticunicellular organism. In varying embodiments, the cell is from amicroalgae. In varying embodiments, the cell is from a cyanobacteria. Insome embodiments, the photosynthetic organism is selected from the groupconsisting of Chlorophyta (green algae), Rhodophyta (red algae),Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae),Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae(chlorarachniophytes), Euglenida (euglenids), Haptophyceae(coccolithophorids), Chrysophyceae (golden algae), Cryptophyta(cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae(coccolithophorids), Bacillariophyta (diatoms), Eustigmatophyceae(eustigmatophytes), Raphidophyceae (raphidophytes), Scenedesmaceae andPhaeophyceae (brown algae). In some embodiments, the photosyntheticorganism is selected from the group consisting of Chlamydomonasreinhardtii, Dunaliella salina, Haematococcus pluvialis, Chlorellavulgaris, Acutodesmus obliquus, and Scenedesmus dimorphus andArthrospira platensis, Arthrospira maxima Anabaena sp.PCC7120,Leptolyngbya sp, Synechocystis sp, and Synechococcus elongates PCC7992.In some embodiments, the cell is a Chlorophyta (green algae) cell. Insome embodiments, the green algae is selected from the group consistingof Chlamydomonas, Dunaliella, Haematococcus, Chlorella, andScenedesmaceae. In some embodiments, the Chlamydomonas is aChlamydomonas reinhardtii. In varying embodiments, the green algae canbe a Chlorophycean, a Chlamydomonas, C. reinhartdii, C. reinhartdii137c, C. reinhardtii cc1690 or a psbA deficient C. reinhartdii strain.In varying embodiments, the cell is from a higher plant selected fromBrassicaceae, Solanaceae, Phaseoleae, Zea and Oryzeae. In someembodiments, the cell comprises at least two (e.g., at least 3, 4, 5, 6,7, 8, 9 or 10) polynucleotides encoding at least two mammalian milk orcolostrum polypeptides. In some embodiments, the at least two mammalianmilk or colostrum polypeptides comprise osteopontin and mammaryassociated serum amyloid A3. In some embodiments, the at least twomammalian milk or colostrum polypeptides comprise lysozyme and mammaryassociated serum amyloid A3. In some embodiments, the one or moremammalian polypeptides further comprises one or more mammalian milk orcolostrum polypeptides selected from immunoglobulins (e.g., IgG1, IgG2,IgA, IgM, IgD), lactoferrin, mammary associated serum amyloid A3,proline rich polypeptide (PRP), growth factors (e.g., transforminggrowth factor (TGF)-β1, TGF-β2, insulin-like growth factor 1(somatomedin C) (IGF-1), IGF-2, epidermal growth factor, heparin-bindingepidermal growth factor-like growth factor, betacellulin), cytokines(e.g., IL-6, IL-1β, IL 1ra) serum albumin, glycomacropeptide, caseinproteins (e.g., β-casein, κ-casein, αs1 casein, αs2-casein andγ-casein), enzymes (e.g., superoxide dismutase, lactoperoxidase,alkaline phosphatase, platelet-activating factor-acetyl hydroxyl ase,lysozyme), 14-3-3 protein zeta chain, 5-oxoprolinase (ATP-hydrolyzing),actin, cytoplasmic 1 (beta-actin), adipose differentiation-relatedprotein, albumin (precursor), aldehyde dehydrogenase (NAD) 2 precursor,ankyrin 3, node of Ranvier (ankyrin G), annexin 1, annexin A2,apolipoprotein A-I, apolipoprotein B, ARP3 (actin-related protein 3,yeast) homolog, ATP synthase, H+ transporting, mitochondrial, F1complex, alpha subunit, beta-2-microglobulin precursor (lactollin);butyrophilin, subfamily 1, member A1; capping protein (actin filament);muscle Z-line, alpha 1; casein kinase 1, alpha 1; coronin, actin bindingprotein, 1A; CD36 antigen [collagen type I receptor, thrombospondinreceptor]; Chitinase-like protein 1 (CLP-1); DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 54; deleted in malignant brain tumors 1; diacylglycerolkinase kappa; endoplasmin precursor (GRP94/GP96); enolase 1; eukaryotictranslation initiation factor 4, gamma 2; fatty acid binding protein,heart-type (MDGI); fetuin; fibrinogen alpha chain; fibrinogen beta chainprecursor; fibrinogen gamma-B chain precursor; gene model 440, (NCBI);glucose regulated protein 58 kD; glutamate receptor, ionotropic, delta1; glutathione S-transferase, mu 1; glyceraldehyde-3-phosphate;dehydrogenase (GAPDH); glycerol-3-phosphate dehydrogenase 2;glycoprotein antigen MGP57/53 (Lactadherin/bP47 protein);glycosylation-dependent cell adhesion molecule 1 (lactophorin/PP3);guanine nucleotide binding protein, beta 2; H3 histone, family 3A; heatshock 70 kDa protein 8; heat shock 70 kD protein 5 (glucose-regulatedprotein); heat shock protein 27; heat shock protein 70 kDa protein 1A;histone 2, H2ab; zinc finger protein 668; hypothetical/unnamed proteinL0051063; IRTA2; isocitrate dehydrogenase 1 (NADP+), soluble; keratin 9;keratin complex 2, basic, gene 6a; keratin, type I cytoskeletal 10; andKIAA1586 protein. In varying embodiments, the one or more mammalianpolypeptides are bioactive. In some embodiments, one or morepolynucleotides encoding the one or more mammalian polypeptides isintegrated into the chloroplast genome or the nuclear genome of thecell, or a cyanobacterial genome, or into a cyanobacterial plasmid. Insome embodiments, the one or more mammalian polypeptides are human milkor colostrum polypeptides. In some embodiments, the one or moremammalian polypeptides are milk or colostrum polypeptides from a mammalselected from the group consisting of human, canine, feline, bovine,porcine, ovine and caprine. In some embodiments, the one or moremammalian polypeptides are milk or colostrum polypeptides. In someembodiments, one or more nucleic acids encoding the one or moremammalian polypeptides selected from osteopontin and lactadherin isintegrated into the chloroplast genome of the cell. In some embodiments,the nucleic acid encoding osteopontin comprises a polynucleotide havingat least about 60% sequence identity, e.g., at least about 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, to SEQ ID NO:7.In some embodiments, the nucleic acid encoding lactadherin comprises apolynucleotide having at least about 60% sequence identity, e.g., atleast about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity, to SEQ ID NO:9. In some embodiments, the nucleic acid encodingcathelicidin-1 comprises a polynucleotide having at least about 60%sequence identity, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% sequence identity, to SEQ ID NO:11. In some embodiments,the one or more mammalian polypeptides are phosphorylated. In varyingembodiments, the one or more mammalian polypeptides are bioactive andphosphorylated at 50% or more, e.g., 60%, 70%, 80%, 90% or more, of theamino acid positions that are phosphorylated in the mammalian peptideexpressed from a mammalian cell. In some embodiments, the one or moremammalian polypeptides comprises bovine osteopontin and the bovineosteopontin is phosphorylated at one or more amino acids comprising S45,S47, S218, S230, S241, S252 and S259, wherein the amino acid positionsare with reference to SEQ ID NO:8 and FIG. 14. In some embodiments, thebovine osteopontin is further phosphorylated at one or more amino acidscomprising S48, T51, S85, S88, T93, T94, S100, S103, S106, S109 andS260, wherein the amino acid positions are with reference to SEQ ID NO:8and FIG. 14. In varying embodiments, the one or more mammalianpolypeptides comprises human osteopontin and the human osteopontin isphosphorylated at one or more amino acids comprising Ser20, Ser22,Ser23, Ser58, Ser60, Ser63, Ser81, Ser84, Ser90, Ser99, Ser102, Ser105,Ser108, Ser111, Thr167, Ser173, Ser177, Ser197, Ser201, Ser206, Ser210,Ser216, Ser236, Ser245, Ser249, Ser252, Ser257, Ser273, Ser285, Ser290,and Ser292, wherein the amino acid positions are with reference to FIGS.3 and 4. In some embodiments, the one or more mammalian polypeptidescomprises canine osteopontin and the canine osteopontin isphosphorylated at one or more amino acids comprising Thr57, Thr60,Ser153, Ser163, Thr164, Ser174, Ser176, Ser198, Ser207, Ser230, Ser233,Ser237, Ser246, Ser282, Ser289, and Ser290, wherein the amino acidpositions are with reference to FIGS. 5 and 6. In some embodiments, theone or more mammalian polypeptides comprises feline osteopontin and thefeline osteopontin is phosphorylated at one or more amino acidscomprising Ser174, Ser176, Ser237, and Ser282, wherein the amino acidpositions are with reference to FIGS. 7 and 8. In some embodiments, theone or more mammalian polypeptides do not disrupt photosyntheticactivity of said organism. In varying embodiments, the one or morepolynucleotides are operably linked to a promoter that promotesexpression in the chloroplast. In varying embodiments, two or morepolynucleotides encoding two or more mammalian milk/colostrumpolypeptides are integrated into the chloroplast genome of the cell. Invarying embodiments, the one or more mammalian polypeptides are retainedor sequestered in the chloroplast of the cell.

In another aspect, provided is a photosynthetic organism comprising oneor more polynucleotides encoding one or more mammalian colostrum or milkproteins is selected from the group consisting of osteopontin,lactoperoxidase, lysozyme, lactadherin, soluble CD14, alpha-lactalbumin,lingual antimicrobial peptide and cathelicidin-1. In varyingembodiments, the photosynthetic organism is a non-vascularphotosynthetic eukaryotic organism. In varying embodiments, thephotosynthetic organism is a photosynthetic unicellular organism. Invarying embodiments, the photosynthetic organism is a cyanobacteria. Insome embodiments, the photosynthetic organism is selected from the groupconsisting of Chlorophyta (green algae), Rhodophyta (red algae),Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae),Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae(chlorarachniophytes), Euglenida (euglenids), Haptophyceae(coccolithophorids), Chrysophyceae (golden algae), Cryptophyta(cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae(coccolithophorids), Bacillafiophyta (diatoms), Eustigmatophyceae(eustigmatophytes), Raphidophyceae (raphidophytes), Scenedesmaceae andPhaeophyceae (brown algae). In some embodiments, the photosyntheticorganism is selected from the group consisting of Chlamydomonasreinhardtii, Dunaliella salina, Haematococcus pluvialis, Chlorellavulgaris, Acutodesmus obliquus, and Scenedesmus dimorphus. In someembodiments, the organism is a Chlorophyta (green algae). In someembodiments, the green algae is selected from the group consisting ofChlamydomonas, Dunaliella, Haematococcus, Chlorella, and Scenedesmaceae.In some embodiments, the Chlamydomonas is a Chlamydomonas reinhardtii.In varying embodiments, the green algae can be a Chlorophycean, aChlamydomonas, C. reinhartdii, C. reinhartdii 137c, or a psbA deficientC. reinhartdii strain. In varying embodiments, the photosyntheticorganism is a higher plant selected from Brassicaceae, Solanaceae,Phaseoleae, Zea and Oryzeae. In some embodiments, the cell comprises atleast two (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10) polynucleotidesencoding at least two mammalian milk or colostrum polypeptides. In someembodiments, at least two mammalian milk or colostrum polypeptidescomprise osteopontin and mammary associated serum amyloid A3. In someembodiments, at least two mammalian milk or colostrum polypeptidescomprise lysozyme and mammary associated serum amyloid A3. In someembodiments, the one or more mammalian polypeptides further comprisesone or more mammalian milk or colostrum polypeptides selected fromimmunoglobulins (e.g., IgG1, IgG2, IgA, IgM, IgD), lactoferrin, mammaryassociated serum amyloid A3, proline rich polypeptide (PRP), growthfactors (e.g., transforming growth factor (TGF)-β1, TGF-β2, insulin-likegrowth factor 1 (somatomedin C) (IGF-1), IGF-2, epidermal growth factor,heparin-binding epidermal growth factor-like growth factor,betacellulin), cytokines (e.g., IL-6, IL-1β, IL 1ra) serum albumin,glycomacropeptide, casein proteins (e.g., β-casein, κ-casein, αs1casein, αs2-casein and γ-casein), enzymes (e.g., superoxide dismutase,lactoperoxidase, alkaline phosphatase, platelet-activatingfactor-acetylhydroxylase, lysozyme), 14-3-3 protein zeta chain,5-oxoprolinase (ATP-hydrolyzing), actin, cytoplasmic 1 (beta-actin),adipose differentiation-related protein, albumin (precursor), aldehydedehydrogenase (NAD) 2 precursor, ankyrin 3, node of Ranvier (ankyrin G),annexin 1, annexin A2, apolipoprotein A-I, apolipoprotein B, ARP3(actin-related protein 3, yeast) homolog, ATP synthase, H+ transporting,mitochondrial, F1 complex, alpha subunit, beta-2-microglobulin precursor(lactollin); butyrophilin, subfamily 1, member A1; capping protein(actin filament); muscle Z-line, alpha 1; casein kinase 1, alpha 1;coronin, actin binding protein, 1A; CD36 antigen [collagen type Ireceptor, thrombospondin receptor]; Chitinase-like protein 1 (CLP-1);DEAD (Asp-Glu-Ala-Asp) box polypeptide 54; deleted in malignant braintumors 1; diacylglycerol kinase kappa; endoplasmin precursor(GRP94/GP96); enolase 1; eukaryotic translation initiation factor 4,gamma 2; fatty acid binding protein, heart-type (MDGI); fetuin;fibrinogen alpha chain; fibrinogen beta chain precursor; fibrinogengamma-B chain precursor; gene model 440, (NCBI); glucose regulatedprotein 58 kD; glutamate receptor, ionotropic, delta 1; glutathioneS-transferase, mu 1; glyceraldehyde-3-phosphate; dehydrogenase (GAPDH);glycerol-3-phosphate dehydrogenase 2; glycoprotein antigen MGP57/53(Lactadherin/bP47 protein); glycosylation-dependent cell adhesionmolecule 1 (lactophorin/PP3); guanine nucleotide binding protein, beta2; H3 histone, family 3A; heat shock 70 kDa protein 8; heat shock 70 kDprotein 5 (glucose-regulated protein); heat shock protein 27; heat shockprotein 70 kDa protein 1A; histone 2, H2ab; zinc finger protein 668;hypothetical/unnamed protein LOC51063; IRTA2; isocitrate dehydrogenase 1(NADP+), soluble; keratin 9; keratin complex 2, basic, gene 6a; keratin,type I cytoskeletal 10; and KIAA1586 protein. In varying embodiments,the one or more mammalian polypeptides are bioactive. In someembodiments, one or more polynucleotides encoding the one or moremammalian polypeptides is integrated into the chloroplast genome of theorganism. In some embodiments, the one or more polynucleotides encodingthe one or more mammalian polypeptides is operably linked to a promoterthat promotes expression in the chloroplast. In some embodiments, theone or more mammalian polypeptides are retained or sequestered in thechloroplast of the organism. In varying embodiments, the one or moremammalian polypeptides comprise a plastid retention sequence comprisinga polynucleotide sequence having at least about 60% sequence identity,e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%sequence identity, to SEQ ID NO:25. In varying embodiments, the one ormore mammalian polypeptides comprise a plastid retention sequencecomprising a polypeptide sequence having at least about 60% sequenceidentity, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or99% sequence identity, to SEQ ID NO:26. In some embodiments, one or morepolynucleotides encoding the one or more mammalian polypeptides isintegrated into the nuclear genome of the organism. In some embodiments,the one or more mammalian polypeptides are human milk or colostrumpolypeptides. In some embodiments, the one or more mammalianpolypeptides are milk or colostrum polypeptides from a mammal selectedfrom the group consisting of human, canine, feline, bovine, porcine,ovine and caprine. In some embodiments, one or more nucleic acidsencoding the one or more mammalian polypeptides selected fromosteopontin and lactadherin is integrated into the chloroplast genome ofthe cell. In some embodiments, the nucleic acid encoding osteopontincomprises a polynucleotide having at least about 60% sequence identity,e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%sequence identity, to SEQ ID NO:7. In some embodiments, the nucleic acidencoding lactadherin comprises a polynucleotide having at least about60% sequence identity, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% sequence identity, to SEQ ID NO:9. In someembodiments, the nucleic acid encoding cathelicidin-1 comprises apolynucleotide having at least about 60% sequence identity, e.g., atleast about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity, to SEQ ID NO:11. In some embodiments, the one or moremammalian polypeptides are phosphorylated. In varying embodiments, theone or more mammalian polypeptides are bioactive and phosphorylated at50% or more, e.g., 60%, 70%, 80%, 90% or more, of the amino acidpositions that are phosphorylated in the mammalian peptide expressedfrom a mammalian cell. In some embodiments, the one or more mammalianpolypeptides comprises bovine osteopontin and .30 the bovine osteopontinis phosphorylated at one or more amino acids comprising S45, S47, S218,S230, S241, S252 and S259, wherein the'amino acid positions are withreference to SEQ ID NO:8 and FIG. 14. In some embodiments, the bovineosteopontin is further phosphorylated at one or more amino acidscomprising S48, T51, S85, S88, T93, T94, S100, S103, S106, S109 andS260, wherein the amino acid positions are with reference to SEQ ID NO:8and FIG. 14. In varying embodiments, the one or more mammalianpolypeptides comprises human osteopontin and the human osteopontin isphosphorylated at one or more amino acids comprising Ser20, Ser22,Ser23, Ser58, Ser60, Ser63, Ser81, Ser84, Ser90, Ser99, Ser102, Ser105,Ser108, Ser111, Thr167, Ser173, Ser177, Ser197, Ser201, Ser206, Ser210,Ser216, Ser236, Ser245, Ser249, Ser252, Ser257, Ser273, Ser285, Ser290,and Ser292, wherein the amino acid positions are with reference to FIGS.3 and 4. In some embodiments, the one or more mammalian polypeptidescomprises canine osteopontin and the canine osteopontin isphosphorylated at one or more amino acids comprising Thr57, Thr60,Ser153, Ser163, Thr164, Ser174, Ser176, Ser198, Ser207, Ser230, Ser233,Ser237, Ser246, Ser282, Ser289, and Ser290, wherein the amino acidpositions are with reference to FIGS. 5 and 6. In some embodiments, theone or more mammalian polypeptides comprises feline osteopontin and thefeline osteopontin is phosphorylated at one or more amino acidscomprising Ser174, Ser176, Ser237, and Ser282, wherein the amino acidpositions are with reference to FIGS. 7 and 8. In varying embodiments,the one or more polynucleotides are operably linked to a promoter thatpromotes expression in the chloroplast. In varying embodiments, two ormore polynucleotides encoding two or more mammalian milk/colostrumpolypeptides are integrated into the chloroplast genome of the organism.In varying embodiments, the one or more mammalian polypeptides areretained or sequestered in the chloroplast of the cell. In someembodiments, the one or more mammalian polypeptides are phosphorylated.For example, proteins produced in chloroplasts or cyanobacteria arepost-translationally modified by phosphorylation with a high level offidelity compared to the same protein produced in other recombinantproduction systems. Colostrum/milk polypeptides produced in chloroplastsor cyanobacteria are characterized by at least 50%, 75%, 85%, 90%, 95%98%, 99% and even up to 100% of the level of bioactivity of the naturalcolostrum-derived counterpart protein. In some embodiments, the one ormore mammalian polypeptides comprise an amino acid sequence thatpromotes secretion from a cell. In some embodiments, the one or moremammalian polypeptides comprise an amino acid sequence that promotesretention on the plasma membrane of a cell. In some embodiments, the oneor more mammalian polypeptides comprise an amino acid sequence thatpromotes protein accumulation. In some embodiments, the one or moremammalian polypeptides do not disrupt photosynthetic activity of saidorganism.

Further provided are methods for producing one or more mammaliancolostrum or milk proteins, comprising culturing a cell or an organismas described above and herein. In some embodiments, the cell or theorganism is grown in the absence of light and in the presence of anorganic carbon source.

Further provided are polynucleotides for expression of colostrum/milkpolypeptides in a chloroplast. In some embodiments, the polynucleotideencoding osteopontin comprises a polynucleotide having at least about60% sequence identity, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% sequence identity, to SEQ ID NO:7. In someembodiments, the polynucleotide encoding lactadherin comprises apolynucleotide having at least about 60% sequence identity, e.g., atleast about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity, to SEQ ID NO:9. In some embodiments, the polynucleotideencoding cathelicidin-1 comprises a polynucleotide having at least about60% sequence identity, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% sequence identity, to SEQ ID NO:11.

Further provided are mammalian osteopontin polypeptides. In varyingembodiments, the osteopontin polypeptide is a bovine osteopontinpolypeptide phosphorylated at one or more amino acids comprising S45,S47, S218, S230, S241, S252 and S259, wherein the amino acid positionsare with reference to SEQ ID NO:8 and FIG. 14. In some embodiments, theosteopontin polypeptide is further phosphorylated at one or more aminoacids comprising S48, T51, S85, S88, T93, T94, S100, S103, S106, S109and S260, wherein the amino acid positions are with reference to SEQ IDNO:8 and FIG. 14. In some embodiments, the bovine osteopontinpolypeptide comprises at least about 60% identity, e.g., at least about65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, toamino acid sequence of SEQ ID NO:8. In varying embodiments, theosteopontin polypeptide is a human osteopontin polypeptide that isphosphorylated at one or more amino acids comprising Ser20, Ser22,Ser23, Ser58, Ser60, Ser63, Ser81, Ser84, Ser90, Ser99, Ser102, Ser105,Ser108, Ser111, Thr167, Ser173, Ser177, Ser197, Ser201, Ser206, Ser210,Ser216, Ser236, Ser245, Ser249, Ser252, Ser257, Ser273, Ser285, Ser290,and Ser292, wherein the amino acid positions are with reference to FIGS.3 and 4. In some embodiments, the human osteopontin polypeptidecomprises at least about 60% identity, e.g., at least about 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, to amino acidsequence of SEQ ID NO:18. In some embodiments, the osteopontinpolypeptide is a canine osteopontin polypeptide that is phosphorylatedat one or more amino acids comprising Thr57, Thr60, Ser153, Ser163,Thr164, Ser174, Ser176, Ser198, Ser207, Ser230, Ser233, Ser237, Ser246,Ser282, Ser289, and Ser290, wherein the amino acid positions are withreference to FIGS. 5 and 6. In some embodiments, the canine osteopontinpolypeptide comprises at least about 60% identity, e.g., at least about65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, toamino acid sequence of SEQ ID NO:20. In some embodiments, theosteopontin polypeptide is a feline osteopontin polypeptide that isphosphorylated at one or more amino acids comprising Ser174, Ser176,Ser237, and Ser282, wherein the amino acid positions are with referenceto

FIGS. 7 and 8. In some embodiments, the feline osteopontin polypeptidecomprises at least about 60% identity, e.g., at least about 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, to amino acidsequence of SEQ ID NO:22.

Further provided are compositions edible by a mammal comprising one ormore populations of cells, one or more populations of organisms and/oran osteopontin polypeptide, as described above and herein. In someembodiments, the composition is selected from a liquid or beverage(e.g., infant formula), a food, a feed, a food supplement, anutraceutical (e.g., a pill). In some embodiments, the composition isselected from the group consisting of a compressed algal cake, an algalpaste and an algal powder. In varying embodiments, the compositions arelyophilized or spray dried. In some embodiments, the photosyntheticorganisms (e.g., algae) are lyophilized or spray-dried prior to theaddition to an edible composition, e.g., a food, beverage or tabletconsumable by a mammal, e.g., a human, a canine, a feline. In someembodiments, the photosynthetic organisms (e.g., algae) are formulatedinto a wet paste prior to the addition to an edible composition, e.g., afood, beverage or tablet consumable by a mammal, e.g., a human, acanine, a feline. In some embodiments, the photosynthetic organisms(e.g., algae) are formulated into a powder to be sprinkled onto or intoan edible composition, e.g., a food, beverage or tablet consumable by amammal, e.g., a human, a canine, a feline. In some embodiments thephotosynthetic organisms (e.g., algae) are blended or mixed into anedible composition, e.g., a food, beverage or tablet consumable by amammal, e.g., a human, a canine, a feline.

Further provided are methods of producing a such compositions edible bya mammal. In varying embodiments, the methods comprise combining two ormore populations of cells or two or more populations of organisms asdescribed above and herein. In some embodiments, the methods comprisecombining two or more of: a population of cells, a population ofcyanobacteria, a population of photosynthetic organisms, and anosteopontin polypeptide, as described above and herein.

Definitions

The term “non-vascular photosynthetic eukaryotic organism” refers to anorganism of the kingdom Planta that does not have xylem or phloem. Theseinclude all species of algae and mosses as well as other photosyntheticorganisms like liverworts.

The term “bioactive” refers to detectable biological activity of apolypeptide, using any assay known in the art to detect the biologicalactivity. The biological activities of the polypeptides described hereinand assays for detecting their biological activity are known in the art.For example, the bioactivity of osteopontin can be measured by theability of osteopontin to adhere to human embryonic 293 cells when inthe presence of the divalent cations, Mg²⁺ or Mn²⁺ but not Ca²⁺ (Hu, etal, J Biol Chem. (1995) 270(17):9917-25). The bioactivity ofmammary-associated serum amyloid (MAA) protein can be determined by thepurified proteins ability to stimulate muc3 production from HT29 cells(Manuell et al., Plant Biotechnol J. (2007) 5(3):402-12). Thebioactivity of lactadherin can be determined by its ability to bind tophosphatidylserine (Otzen, et al., Biochim Biophys Acta. (2012)1818(4):1019-27). Cathelicidin-1 activity can be determined using anantimicrobial assay and measuring luminescence. See, e.g., Sue, et al.Infect Immun. 2000 68(5) 2748-2755.

The terms “identical” or percent “identity,” and variants thereof in thecontext of two or more polynucleotide or two or more amino acidsequences, refer to two or more sequences or subsequences that are thesame. Sequences are “substantially identical” if they have a specifiedpercentage of nucleic acid residues or amino acid residues that are thesame (i.e., at least 60% identity, optionally at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a referencesequence (e.g., SEQ ID NOs: 1-24) over a specified region (or the wholereference sequence when not specified)), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using any sequence comparison algorithm known in the art (GAP,BESTFIT, BLAST, Align, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis.), Karlin and Altschul Proc. Natl. Acad. Sci. (U.S.A.)87:2264-2268 (1990) set to default settings, or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995-2014). Provided are polynucleotides improved forexpression in photosynthetic (e.g., algal) host cells that aresubstantially identical to the polynucleotides of SEQ ID NOs: 1, 3, 5,7, 9, 11, 17, 19, 21 and 23. Provided are polypeptides expressed inphotosynthetic (e.g., algal) host cells that are substantially identicalto the polypeptides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16,17, 20, 22 and 24. Optionally, the identity exists over a region that isat least about 50, 100, 150, 200, 250, 300 amino acids in length, ormore preferably over a region that is 100, 200, 300, 400, 500, 600, 800,1000, or more, nucleic acids in length, or over the full-length of thesequence.

The term “conservatively modified variations” refers to individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence, where thealterations result in the substitution of an amino acid with achemically similar amino acid; and the alterations, deletions oradditions do not alter the structure, function and/or immunogenicity ofthe sequence. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates examples of colostrum/milk proteins and theirbioactivities

FIG. 2 illustrates a polynucleotide sequence with altered codons forimproved expression of bovine osteopontin from the chloroplast genomeand the corresponding amino acid sequence.

FIGS. 3 illustrates a polynucleotide sequence with altered codons forimproved expression of human osteopontin from the chloroplast genome andthe corresponding amino acid sequence. Optional N-terminal STREP-TAG® isunderlined.

FIG. 4 illustrates the amino acids that are phosphorylated on the humanvariant of osteopontin expressed from a chloroplast genome. Thephosphorylated residues include Ser²⁰, Ser²², Ser²³, Ser⁵⁸, Ser⁶⁰,Ser⁶³, Ser⁸¹, Ser⁸⁴, Ser⁹⁰, Ser⁹⁹, Ser¹⁰², Ser¹⁰⁵, Ser¹⁰⁸, Ser¹¹¹,Thr¹⁶⁷, Ser¹⁷³, Ser¹⁷⁷, Ser¹⁹⁷, Ser²⁰¹, Ser²⁰⁶, Ser²¹⁰, Ser²¹⁶, Ser²³⁶,Ser²⁴⁵, Ser²⁴⁹, Ser²⁵², Ser²⁵⁷, Ser²⁷³, Ser²⁸⁵, Ser²⁹⁰, and Ser²⁹².Amino acid position numbers are with reference to FIGS. 3 and 4. Seealso, Christensen et al. Biochem J. 2005 Aug. 15; 390(Pt 1): 285-292.

FIG. 5 illustrates a polynucleotide sequence with altered codons forimproved expression of canine osteopontin from the chloroplast genomeand the corresponding amino acid sequence. Optional N-terminal FLAG-tagis underlined.

FIG. 6 illustrates the amino acids that are phosphorylated on the caninevariant of osteopontin expressed from a chloroplast genome. Thephosphorylated residues include Thr⁵⁷, Thr⁶⁰, Ser¹⁵³, Ser¹⁶³, Thr¹⁶⁴,Ser¹⁷⁴, Ser¹⁷⁶, Ser¹⁹⁸, Ser²⁰⁷, Ser²³⁰, Ser²³³, Ser²³⁷, Ser²⁴⁶, Ser²⁸²,Ser²⁸⁹, and Ser²⁹⁰. Amino acid position numbers are with reference toFIGS. 5 and 6.

FIG. 7 illustrates a polynucleotide sequence with altered codons forimproved expression of feline osteopontin from the chloroplast genomeand the corresponding amino acid sequence. Optional N-terminal FLAG-tagis underlined.

FIG. 8 illustrates the amino acids that are phosphorylated on the felinevariant of osteopontin expressed from a chloroplast genome. Thephosphorylated residues include Ser¹⁷⁴, Ser¹⁷⁶, Ser²³⁷, and Ser²⁸².Amino acid position numbers are with reference to FIGS. 7 and 8.

FIGS. 9A-B illustrate a polynucleotide sequence with altered codons forimproved expression of lactadherin from the chloroplast genome and thecorresponding amino acid sequence.

FIG. 10 illustrates a polynucleotide sequence with altered codons forimproved expression of cathelicidin-1 from the chloroplast genome andthe corresponding amino acid sequence.

FIG. 11 illustrates a Western blot analysis of transgenic algae strainswhose chloroplast genome has been transformed with a flag taggedosteopontin gene. Westerns probed with an anti-flag antibody. Lane 1Negative control. Lanes 2-6: Independent transgenic strains.

FIG. 12 illustrates a Western blot analysis of transgenic algae strainswhose chloroplast genome has been transformed with a flag taggedlactadherin gene. Westerns probed with an anti-flag antibody. Lane 1Negative control. Lanes 2-5: Independent transgenic strains.

FIG. 13 illustrates a Western blot analysis of transgenic algae strainswhose chloroplast genome has been transformed with a flag taggedcathelicidin-1 gene. Westerns probed with an anti-flag antibody. Lane 1Negative control. Lanes 2-5: Independent transgenic strains.

FIG. 14A illustrates a mass spectrometry analysis of purifiedchloroplast expressed bovine osteopontin. FIG. 14B summarizes mass specidentification of phosphorylation sites of bovine osteopontin expressedfrom a chloroplast. Localized phosphorylation sites on chloroplastexpressed bovine osteopontin include S45, S47, S218, S230, S241, S252and S259. Ambiguous phosphorylation sites on chloroplast expressedbovine osteopontin include S48, T51, S84, S88, T93, T94, S100, S103,S106, S109 and S260. Amino acid position numbers are with reference toSEQ ID NO:8.

FIG. 15 illustrates cell adhesion bioactivity assay for bovineosteopontin expressed from the chloroplast genome. Purified chloroplastbovine osteopontin was coated on a microtiter plate and cell adhesionassays performed. 293 kidney cells in the presence of 2 mM Mg²⁺ wereincubated in microtiter plates coated with chloroplast produced bovineosteopontin. Unbound cells were washed away and 1004, culture medium wasadded to each well. 10 μL of wst-8 reagent (cell identification reagent)was added to each well and plates were incubated for 1 hour. Followingincubation the absorbance was measured at 450 nm and compared to astandard curve to determine the percent of cells that bound the algalproduced chloroplast osteopontin in each well. Chloroplast-expressedbovine osteopontin was bound by up to 40% of the cells (170 nMosteopontin).

FIG. 16 illustrates bioactivity of lactadherin expressed from thechloroplast genome in binding to phosphatidylserine.

FIG. 17 illustrates a Western blot showing the accumulation of bovineosteopontin protein in the cyanobacteria Anabaena. Lane 1 containswild-type anabaena. Lanes 2 and 3 contain independent anabaenatransformations transformed with an osteopontin gene containing achloroplast codon bias. Lanes 4 and 5 contain independent anabaenatransformations that were transformed with an osteopontin gene coded ina nuclear genome bias. Lane 6 contains a transgenic Chlamydomonasreinhardtii strain accumulating osteopontin that is serving as apositive control. Transgenic protein was detected using a rabbitpolyclonal antibody directed against bovine osteopontin.

FIG. 18 illustrates a Western blot showing the accumulation of bovineosteopontin in the cyanobacteria Leptolyngbya and Synechocystis 6803.Lane 1 contains a transgenic Chlamydomonas reinhardtii strainaccumulating bovine osteopontin that is serving as a positive control.Lane 2 contains a Protein Ladder to serve as a size standard. Lane 3contains wild-type Leptolyngbya. Lane 4 and 5 contain Leptolyngbyatransformed with a bovine osteopontin gene that contains a nuclear codonbias. Lane 6 contains wild-type Synechocystis 6803. Lane 7 and Lane 8contain independent transgenic Synechocystis 6803 strains transformedwith a bovine osteopontin that was coded in a chloroplast codon bias.Lane 9 and 10 contain transgenic Synechocystis 6803 transformed with abovine osteopontin gene coded in a nuclear codon bias.

FIG. 19 illustrates a Western blot showing the accumulation of MAA inthe cyanobacteria Anabaena. Lane 1 contains a transgenic Chlamydomonasreinhardtii strain transformed with the mammary-associated serum amyloidA3 (MAA) gene. Lane 2 contains wild-type anabaena. Lane 3 contains atransgenic anabaena strain transformed with a MAA gene that was coded ina nuclear codon bias. Westerns were detected using a polyclonal antibodydirected against the MAA protein.

FIG. 20 illustrates a Western blot showing the accumulation of MAA inSynechococcus elongates 7942. Lane 1 contains wild-type Synechococcuselongates 7942. Lane 2 contains a transgenic Synechococcus elongatus7942 transformed with the MAA gene that has not been induced for proteinaccumulation. Lane 3 contains a transgenic Synechococcus elongatus 7942transformed with the MAA gene that has been induced to accumulate MAAprotein.

FIG. 21 illustrates Western blots showing the accumulation of MAA andosteopontin both expressed in the chloroplast genome. The lanes containthe following: Lane 1: Negative Control, Lane 2: A Protein Standard,Lane 3 Soluble protein from a transgenic strain of C. reinhartdiiexpressing only osteopontin from the chloroplast genome, Lane 4: Totalfrom a transgenic strain of C. reinhartdii expressing only osteopontinfrom the chloroplast genome, Lane 5: Soluble protein from a transgenicstrain expressing both osteopontin and MAA from the chloroplast genome,Lane 6: Total protein from a transgenic strain expressing bothosteopontin and MAA from the chloroplast genome, Lane 7: Soluble proteinfrom a transgenic strain of C. reinhartdii expressing only MAA from thechloroplast genome, Lane 8: Total from a transgenic strain of C.reinhartdii expressing only MAA from the chloroplast genome. The topWestern is probed with an antibody against the M AA protein and thebottom Western was probed with an antibody against the flag tag of theosteopontin protein.

DETAILED DESCRIPTION 1. Introduction

Described herein are compositions and processes to produce bioactivecolostrum and/or milk proteins for health and nutrition purposes usingchloroplast-engineered photosynthetic organisms (e.g., algae) as both aproduction and delivery vehicle. The organisms and processes describedherein provide an alternative system and organisms for lower cost andlarge-scale production of singular and/or tailored mixtures/combinationsof orally active colostrum and milk bioactives in an orally availableform (e.g., edible algae). In varying embodiments, two or morecolostrum/milk polypeptides can be expressed from the chloroplasts ofthe same organism.

Genes encoding bioactive colostrum/milk proteins have altered codons forexpression from the chloroplast genomes of edible photosyntheticorganisms (e.g., for example, higher plants, algae, microalgae,including Chlorophyta , e.g., Chlamydomonas reinhardtii) or from thegenome or plasmid of cyanobacteria). Illustrative colostrum/milkproteins include without limitation mammary associated serum amyloid A3,lactoperoxidase, lactoferrin, osteopontin, lysozyme, alpha-lactalbumin,lactadherin, soluble CD14, cathelicidin-1, and lingual antimicrobialpeptide (FIG. 1). The colostrum/milk genes can be integrated into andexpressed from the chloroplast genomes of photosynthetic organisms.Expression and bioactivity can be confirmed using methods known in theart.

Production and/or delivery of colostrum/milk polypeptides in ediblephotosynthetic organisms finds use, e.g., in human and mammal health andnutrition; prophylaxis and treatment for enteric infection; prophylaxisand/or treatment of gastric, intestinal, or bowel inflammation;improving nutrient uptake efficiency; improving bone strength; foodpreservation and processing; cosmetics preservation; odor treatment andneutralization; oral hygiene; acne treatment; and topical and oralantibacterial, antiviral, and/or antimicrobial therapy.

2. Colostrum/Milk Polynucleotides and Polypeptides

Photosynthetic eukaryotic organisms have one or more polynucleotidesencoding one or more mammalian colostrum/milk polypeptides areintegrated into the chloroplast genome. In varying embodiments, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, polynucleotides encoding mammaliancolostrum/milk polypeptides are independently integrated into thechloroplast genome of a photosynthetic organism.

Illustrative mammalian colostrum/milk polypeptides for expression inphotosynthetic organisms (e.g. chlorophyta, e.g., Chlamydomonas), andchloroplast and cells thereof, are described above and herein. See,e.g., Smolenski, et al., J Proteome Res. 2007 Jan.; 6(1):207-15; Boudryand Thewis, Bulletin UASVM Animal Science and Biotechnologies (2009) 66(1-2); Chatterton, et al., Intl Journal of Biochemistry & Cell Biology45 (2013) 1730-1747; Lis, et al., Postepy Hig Med Dosw (2013) 67:529-547; and Artym, et al., Postepy Hig Med Dosw (2013) 67: 800-816. Invarying embodiments, the one or more colostrum/milk polypeptides arewhey proteins (e.g., alpha-lactalbumim, beta-lactoglobulin, osteopontin,lactoferrin and/or immunoglobulins). The expressed mammaliancolostrum/milk polypeptides, and chloroplasts, cells and photosyntheticorganisms comprising the polypeptides, can be used as and incompositions edible by a mammal (e.g., having both nutritional andtherapeutic value).

In varying embodiments, the milk/colostrum polypeptides are human,non-human primate, bovinae (e.g., cow, bison), ovine, caprine, camelid,human, canine, feline, equine, marsupial, or from any other mammal ofinterest. The polynucleotide and polypeptide sequences of mammalianhomologs of milk/colostrum polypeptides are known in the art. Forexample, the GenBank Ref. Seq. Accession Nos. for osteopontinpolypeptide homologs are NP_000573.1 (human), XP_003434072.1 (canine),XP_003985233.1 (feline), and NP 776612.1 (bovine). For example,mammalian milk/colostrum proteins (e.g., osteopontin, e.g., from ahuman, canine, or feline) can be produced in a photosynthetic organism(e.g., algae) and subsequently lyophilized and sprinkled onto a food orinto a beverage consumable by the mammal (e.g., human, canine, orfeline, respectively). In another example, mammalian milk/colostrumproteins (e.g., from a human, canine, feline or equine) produced in aphotosynthetic organism (e.g., algae) can be formulated into a wet pasteand delivered orally to the mammal (e.g., to the human, canine, felineor equine), e.g., using a syringe. In another example, lyophilized,freeze-dried or spray-dried photosynthetic organisms (e.g., algae)comprising mammalian milk/colostrum polypeptides can be re-suspended inwater for oral delivery to the mammal (e.g., to the human, canine,feline or equine), e.g., using a syringe. In another example,lyophilized, freeze-dried, spray-dried or powdered photosyntheticorganisms (e.g., algae) comprising mammalian milk/colostrum polypeptidescan be sprayed onto or mixed or blended into a food, feed or beverageedible by a mammal (e.g., to the human, canine, feline or equine), e.g.,sprayed onto kibble for a non-human mammal.

Polynucleotides encoding one or more milk/colostrum polypeptides, orimmunogenic fragments thereof, can be altered for improved expression ina photosynthetic (e.g., algal) host cells. For example, codons in thewild-type polynucleotides encoding one or more milk/colostrumpolypeptides rarely used by the photosynthetic (e.g., algal) host cellcan be replaced with a codon coding for the same or a similar amino acidresidue that is more commonly used by the photosynthetic (e.g., algal)host cell (i.e., employing algal chloroplast codon bias), therebyallowing for more efficient expression of the milk/colostrum polypeptideand higher yields of the expressed milk/colostrum polypeptide in thephotosynthetic host, in comparison to expression of the milk/colostrumpolypeptide from the wild-type polynucleotide. Methods for alteringpolynucleotides for improved expression in a photosynthetic (e.g.,algal) host cell, particularly in a Chlamydomonas reinhardtii host cell,are known in the art and described in, e.g., Franklin et al (2002) PlantJ30:733-744; F1 etcher, et al., Adv Exp Med Biol. (2007) 616:90-8;Heitzer, et al., Adv Exp Med Biol. (2007) 616:46-53; Rasala andMayfield, Bioeng Bugs. (2011) 2(1):50-4; Rasala, et al, Plant BiotechnolJ. (2010) 8(6):719-33; Wu, et al., Bioresour Technol. (2011)102(3):2610-6; Morton, J Mol Evol. (1993) 37(3):273-80; Morton, J MolEvol. (1996) 43(1):28-31; and Morton, J Mol Evol. (1998) 46(4):449-59.

In various embodiments, polynucleotide sequences encoding milk/colostrumpolypeptides can be improved for expression in photosynthetic organisms(e.g., algae) by changing codons that are not common in the algae hostcell (e.g., used less than ˜20% of the time). A codon usage database ofuse is found at kazusa.or.jp/codon/. For improved expression ofpolynucleotide sequences encoding milk/colostrum polypeptides in C.reinhardtii host cells, codons rare or not common to the chloroplast ofC. reinhartdii in the native milk/colostrum nucleic acid sequences arereduced or eliminated. A representative codon table summarizing codonusage in the C. reinhartdii chloroplast is found on the interne atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3055.chloroplast. Invarious embodiments, preferred or more common codons for amino acidresidues in C. reinhartdii are as follows:

Preferred codons for improved Amino Acid Residue expression in algae AlaGCT, GCA Arg CGT Asn AAT Asp GAT Cys TGT Gln CAA Glu GAA Gly GGT Ile ATTHis CAT Leu TTA Lys AAA Met ATG Phe TTT Pro CCA Ser TCA Thr ACA, ACT TrpTGG Tyr TAT Val GTT, GTA STOP TAA

In certain instances, less preferred or less common codons forexpression in an algal host cell can be included in a polynucleotidesequence encoding a milk/colostrum polypeptide, for example, to avoidsequences of multiple or extended codon repeats, or sequences of reducedstability (e.g., extended A/T-rich sequences), or having a higherprobability of secondary structure that could reduce or interfere withexpression efficiency. In various embodiments, the polynucleotidesequence can be synthetically prepared. For example, the desired aminoacid sequence of a milk/colostrum polypeptide as described herein can beentered into a software program with algorithms for determining codonusage for a photosynthetic (e.g., algal) host cell. Illustrativesoftware includes GeneDesigner available from DNA 2.0, on the internetat dna20.com/genedesigner2.

In varying embodiments, the polypeptides are phosphorylated.Chloroplast-expressed gene products provide a distinct advantage overthose encoded in the nuclear genome, particularly in the case in whichphosphorylation contributes to the biologic activity of the end productprotein. In varying embodiments, mammalian polypeptides expressed fromthe chloroplasts of photosynthetic organisms are phosphorylated andbioactive. In varying embodiments, the pattern of phosphorylation of themammalian polypeptide expressed from the chloroplast is different isdifferent from the pattern of phosphorylation of the mammalianpolypeptide expressed from a mammalian cell. In varying embodiments, theone or more mammalian polypeptides are bioactive and phosphorylated at50% or more, e.g., 60%, 70%, 80%, 90% or more, of the amino acidpositions that are phosphorylated in the mammalian peptide expressedfrom a mammalian cell. In some embodiments, the one or more mammalianpolypeptides comprises bovine osteopontin and the bovine osteopontin isphosphorylated at one or more amino acids comprising S45, S47, S218,S230, S241, S252 and S259, wherein the amino acid positions are withreference to SEQ ID NO:8 and FIG. 14. In some embodiments, the bovineosteopontin is further phosphorylated at one or more amino acidscomprising S48, T51, S85, S88, T93, T94, S100, S103, S106, S109 andS260, wherein the amino acid positions are with reference to SEQ ID NO:8and FIG. 14. In varying embodiments, the one or more mammalianpolypeptides comprises human osteopontin and the human osteopontin isphosphorylated at one or more amino acids comprising Ser20, Ser22,Ser23, Ser58, Ser60, Ser63, Ser81, Ser84, Ser90, Ser99, Ser102, Ser105,Ser108, Ser111, Thr167, Ser173, Ser177, Ser197, Ser201, Ser206, Ser210,Ser216, Ser236, Ser245, Ser249, Ser252, Ser257, Ser273, Ser285, Ser290,and Ser292, wherein the amino acid positions are with reference to FIGS.3 and 4. In some embodiments, the one or more mammalian polypeptidescomprises canine osteopontin and the canine osteopontin isphosphorylated at one or more amino acids comprising Thr57, Thr60,Ser153, Ser163, Thr164, Ser174, Ser176, Ser198, Ser207, Ser230, Ser233,Ser237, Ser246, Ser282, Ser289, and Ser290, wherein the amino acidpositions are with reference to FIGS. 5 and 6. In some embodiments, theone or more mammalian polypeptides comprises feline osteopontin and thefeline osteopontin is phosphorylated at one or more amino acidscomprising Ser174, Ser176, Ser237, and Ser282, wherein the amino acidpositions are with reference to FIGS. 7 and 8.

In varying embodiments, the polynucleotide sequences encoding themammalian milk/colostrum polypeptides can further encode a sequence thatpromotes protein accumulation. Protein accumulation amino acid sequencesare known in the art and find use.

The psbA promoter and untranslated regions (UTRs) supports high levelsof recombinant protein accumulation in C. reinhartdii. Accordingly, invarying embodiments, the polynucleotide encoding one or morecolostrum/milk polypeptides is operably linked to a polynucleotideencoding a psbA promoter and 5′UTR, an atpA promoter and 5′ UTR, or apsbD promoter and 5′ UTR. In varying embodiments, the psbA promoter and5′ UTR, an atpA promoter and 5′ UTR, a TufA promoter and 5′ UTR, a atpBpromoter and 5′ UTR, or a psbD promoter and 5′ UTR. is upstream of thepolynucleotide encoding the one or more colostrum/milk polypeptides. Inother embodiments the polynucleotide encoding one or more colostrum/milkpolypeptides is operably linked to a polynucleotide encoding a psbA 3′UTR or a rbcL 3′ UTR that is downstream of the nucleotide sequenceencoding one or more colostrum/milk polypeptides. See, e.g., U.S. PatentPublication No. 2012/0309939.

In varying embodiments, the chloroplasts of photosynthetic (e.g., algal)host cells are transformed, e.g., by homologous recombinationtechniques, to contain and stably express one or more polynucleotidesencoding one or more milk/colostrum polypeptides, as described herein,integrated into the chloroplast genome.

Transformation of the chloroplasts of photosynthetic (e.g., algal) hostcells can be carried out according to techniques well known to thosepersons skilled in the art. Examples of such techniques include withoutlimitation electroporation, particle bombardment, cytoplasmic or nuclearmicroinjection, gene gun. See, e.g., FIG. 2 of WO 2012/170125.

3. Photosynthetic Organisms

The colostrum/milk polypeptides can be integrated into and expressedfrom the chloroplast genome of a eukaryotic photosynthetic organism. Thecolostrum/milk polypeptides can be integrated into the genome orexpressed from a plasmid of cyanobacteria. Photosynthetic organismsuseful for the expression of colostrum/milk polypeptides include,without limitation, higher plant chloroplasts, algae (includingmicroalgae), and cyanobacteria. In varying embodiments, thephotosynthetic organism can be eukaryotic (e.g., higher plants andalgae, including microalgae and macroalgae) or prokaryotic (e.g.,cyanobacteria). Plants of interest include vascular plant (e.g., abrassica, corn, soybean, tobacco, rice, etc), and non-vascular plants(e.g., algae, including microalgae, and mosses). Embodiments ofphotosynthetic organisms are described above and herein.

In varying embodiments, the chloroplast, nucleus, cell and/or organismis a microalgae. Illustrative and additional microalgae species ofinterest include without limitation, Achnanthes orientalis, Agmenellum,Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea,Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Carteria, Chaetoceros gracilis, Chaetocerosmuelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlamydomonassp., Chlamydomonas reinhartdii, Chlorella anitrata, ChlorellaAntarctica, Chlorella aureoviridis, Chlorella candida, Chlorellacapsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorellaemersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorellaglucotropha, Chlorella infusionum, Chlorella infusionum var. actophila,Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorellalobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorellaluteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens,Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorellanocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii,Chlorella protothecoides, Chlorella protothecoides var. acidicola,Chlorella regularis, Chlorella regularis var. minima, Chlorellaregularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila,Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorellasimplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica,Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris,Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgarisvar. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgarisvar. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorellavulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorellazofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcuminfusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsasp., Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana,Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Naimochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschiadissipata, Nitzschia ihrstulum, Nitzschia hantzschiana, Nitzschiainconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschiapusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis,Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoriasp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus,Phormidium, Platymonas sp., Plezirochrysis carterae, Pleurochlysisdentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus,Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp.,Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica,Thalassiosira weissflogii, and Viridiella fridericiana.

In varying embodiments, the chloroplast, cell and/or organism is from ahigher plant or vascularized plant. Illustrative and additional plantspecies of interest include without limitation, Brassicaceae (broccoli,cabbage, cauliflower, kale), Solanaceae (e.g., tomato, potato, tobacco),Phaseoleae (e.g., soybean), Zea (e.g., corn) and Oryzeae (e.g., rice).

4. Methods of Producing

Recombinant expression of proteins from heterologous polynucleotidesincorporated into the chloroplast genome of a photosynthetic (e.g.,algal) host cell, particularly a Chlorophyta (green algae) host cell ofthe genus Chlamydomonas, in particular Chlamydomonas reinhartdii, isknown in the art, finds use, and is described in numerous publications,including, e.g., in Rasala and Mayfield, Bioeng Bugs. (2011) 2(1):50-4;Rasala, et al., Plant Biotechnol J. (2011) May 2, PMID 21535358;Coragliotti, et al., Mol Biotechnol. (2011) 48(1):60-75; Specht, et al.,Biotechnol Lett. (2010) 32(10):1373-83; Rasala, et al., Plant BiotechnolJ. (2010) 8(6):719-33; Mulo, et al., Biochim Biophys Acta. (2011) May 2,PMID:21565160; and Bonente, et al., Photosynth Res. (2011) May 6,PMID:21547493; U.S. Patent Publication No. 2012/0309939; U.S. PatentPublication No. 2010/0129394; and Intl. Publication No. WO 2012/170125.All of the foregoing references are incorporated herein by reference intheir entirety for all purposes.

a. Culturing of Cells or Organisms

Techniques for culturing of microalgae and cyanobacteria and vascularplants for expression of recombinant polypeptides are known in the artand can be used for the production of milk/colostrum polypeptides. Thephotosynthetic organism containing the recombinant polynucleotidesencoding one or more colostrum/milk polypeptides can be grown underconditions which permit photosynthesis, however, this is not arequirement (e.g., a host organism may be grown in the absence oflight). In some instances, the host organism may be genetically modifiedin such a way that its photosynthetic capability is diminished ordestroyed. In growth conditions where a host organism is not capable ofphotosynthesis (e.g., because of the absence of light and/or geneticmodification), typically, the organism will be provided with thenecessary nutrients to support growth in the absence of photosynthesis.For example, a culture medium in (or on) which an organism is grown, maybe supplemented with any required nutrient, including an organic carbonsource, nitrogen source, phosphorous source, vitamins, metals, lipids,nucleic acids, micronutrients, and/or an organism-specific requirement.Organic carbon sources include any source of carbon which the hostorganism is able to metabolize including, but not limited to, acetate,simple carbohydrates (e.g., glucose, sucrose, and lactose), complexcarbohydrates (e.g., starch and glycogen), proteins, and lipids. One ofskill in the art will recognize that not all organisms will be able tosufficiently metabolize a particular nutrient and that nutrient mixturesmay need to be modified from one organism to another in order to providethe appropriate nutrient mix.

Organisms can be grown on a defined minimal medium (for example, highsalt medium (HSM), modified artificial sea water medium (MASM), or F/2medium) with light as the sole energy source. In other instances, theorganism can be grown in a medium (for example, tris acetate phosphate(TAP) medium), and supplemented with an organic carbon source.

Organisms, such as algae, can grow naturally in fresh water or marinewater. Culture media for freshwater algae can be, for example, syntheticmedia, enriched media, soil water media, and solidified media, such asagar. Various culture media have been developed and used for theisolation and cultivation of fresh water algae and are described inWatanabe, M. W. (2005). Freshwater Culture Media. In R. A. Andersen(Ed.), Algal Culturing Techniques (pp. 13-20). Elsevier Academic Press,2005. Culture media for marine algae can be, for example, artificialseawater media or natural seawater media. Guidelines for the preparationof media are described in Harrison, P. J. and Berges, J. A. (2005).Marine Culture Media. In R. A. Andersen (Ed.), Algal CulturingTechniques (pp. 21-33). Elsevier Academic Press, 2005.

Culturing techniques for algae are well known to one of skill in the artand are described, for example, in Freshwater Culture Media. In R. A.Andersen (Ed.), Algal Culturing Techniques. Elsevier Academic Press,2005. See also, Richmond and Hu, Handbook of Microalgal Culture: AppliedPhycology and Biotechnology, Wiley-Blackwell; 2nd edition (Jun. 4,2013). In varying embodiments, algae can be grown in a bioreactor or afermenter using either sunlight or reduced carbon as an energy source.

Chlamydomonas sp., Scenedesmus sp., and Chlorella sp. are illustrativealgae that can be cultured as described herein and can grow under a widearray of conditions.

One organism that can be cultured as described herein is a commonly usedlaboratory species C. reinhartdii. Cells of this species are haploid,and can grow on a simple medium of inorganic salts, using photosynthesisto provide energy. This organism can also grow in total darkness ifacetate is provided as a carbon source. C. reinhartdii can be readilygrown at room temperature under standard fluorescent lights. Inaddition, the cells can be synchronized by placing them on a light-darkcycle. Other methods of culturing C. reinhartdii cells are known to oneof skill in the art.

b. Introduction of Polynucleotide into a Host Organism or Cell

To generate a genetically modified host cell, a polynucleotide, or apolynucleotide cloned into a vector, is introduced stably or transientlyinto a host cell, using established techniques, including, but notlimited to, electroporation, biolistic, calcium phosphate precipitation,DEAE-dextran mediated transfection, and liposome-mediated transfection.For transformation, a polynucleotide of the present disclosure willgenerally further include a selectable marker, e.g., any of severalwell-known selectable markers such as restoration of photosynthesis, orkanamycin resistance or spectinomycin resistance.

A polynucleotide or recombinant nucleic acid molecule described herein,can be introduced into a cell (e.g., alga cell) using any method knownin the art. A polynucleotide can be introduced into a cell by a varietyof methods, which are well known in the art and selected, in part, basedon the particular host cell. For example, the polynucleotide can beintroduced into a cell using a direct gene transfer method such aselectroporation or microprojectile mediated (biolistic) transformationusing a particle gun, or the “glass bead method,” or by pollen-mediatedtransformation, liposome-mediated transformation, transformation usingwounded or enzyme-degraded immature embryos, or wounded orenzyme-degraded embryogenic callus (for example, as described inPotrykus, Ann. Rev. Plant. Physiol. Plant Mol. Biol. 42:205-225, 1991).

As discussed above, microprojectile mediated transformation can be usedto introduce a polynucleotide into a cell (for example, as described inKlein et al., Nature 327:70-73, 1987). This method utilizesmicroprojectiles such as gold or tungsten, which are coated with thedesired polynucleotide by precipitation with calcium chloride,spermidine or polyethylene glycol. The microprojectile particles areaccelerated at high speed, into a cell using a device such as theBIOLISTIC PD-1000 particle gun (BioRad; Hercules Calif.). Methods forthe transformation using biolistic methods are well known in the art(for example, as described in Christou, Trends in Plant Science1:423-431, 1996). Microprojectile mediated transformation has been used,for example, to generate a variety of transgenic plant species,including cotton, tobacco, corn, hybrid poplar and papaya. Importantcereal crops such as wheat, oat, barley, sorghum and rice also have beentransformed using microprojectile mediated delivery (for example, asdescribed in Duan et al., Nature Biotech. 14:494-498, 1996; andShimamoto, Curr. Opin. Biotech. 5:158-162, 1994). The transformation ofmost dicotyledonous plants is possible with the methods described above.Transformation of monocotyledonous plants also can be transformed using,for example, biolistic methods as described above, protoplasttransformation, electroporation of partially permeabilized cells,introduction of DNA using glass fibers, and the glass bead agitationmethod.

The basic techniques used for transformation and expression inphotosynthetic microorganisms are similar to those commonly used for E.coli, Saccharomyces cerevisiae and other species. Transformation methodscustomized for photosynthetic microorganisms, e.g., the chloroplast of astrain of algae, are known in the art. These methods have been describedin a number of texts for standard molecular biological manipulation (seePacker & Glaser, 1988, “Cyanobacteria”, Meth. Enzymol., Vol. 167;Weissbach & Weissbach, 1988, “Methods for plant molecular biology,”Academic Press, New York, Green and Sambrook, Molecular Cloning, ALaboratory Manual, 4th Ed., Cold Spring Harbor Press, (2012); and ClarkM S, 1997, Plant Molecular Biology, Springer, N.Y.). These methodsinclude, for example, biolistic devices (See, for example, Sanford,Trends In Biotech. (1988) .delta.: 299-302, U.S. Pat. No. 4,945,050;electroporation (Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824-5828); use of a laser beam, electroporation, microinjection or anyother method capable of introducing DNA into a host cell.

Plastid transformation is a routine and well known method forintroducing a polynucleotide into a plant cell chloroplast (see U.S.Pat. Nos. 5,451,513, 5,545,817, and 5,545,818; WO 95/16783; McBride etal., Proc. Natl. Acad. Sci., USA 91:7301-7305, 1994). In someembodiments, chloroplast transformation involves introducing regions ofchloroplast DNA flanking a desired nucleotide sequence, allowing forhomologous recombination of the exogenous DNA into the targetchloroplast genome. In some instances one to 1.5 kb flanking nucleotidesequences of chloroplast genomic DNA may be used. Using this method,point mutations in the chloroplast 16S rRNA and rps12 genes, whichconfer resistance to spectinomycin and streptomycin, can be utilized asselectable markers for transformation (Svab et al., Proc. Natl. Acad.Sci., USA 87:8526-8530, 1990), and can result in stable homoplasmictransformants, at a frequency of approximately one per 100 bombardmentsof target leaves.

In some embodiments, an alga is transformed with one or morepolynucleotides which encode one or more milk/colostrum polypeptides, asdescribed herein. In one embodiment, a transformation may introduce anucleic acid into a plastid of the host alga (e.g., chloroplast). Inanother embodiment, a transformation may introduce a second nucleic acidinto the chloroplast genome of the host alga. In still anotherembodiment, a transformation may introduce two protein coding regionsinto the plastid genome on a single gene, or may introduced two genes ona single transformation vector.

Transformed cells can be plated on selective media followingintroduction of exogenous nucleic acids. This method may also compriseseveral steps for screening. A screen of primary transformants can beconducted to determine which clones have proper insertion of theexogenous nucleic acids. Clones which show the proper integration may bepropagated and re-screened to ensure genetic stability. Such methodologyensures that the transformants contain the genes of interest. In manyinstances, such screening is performed by polymerase chain reaction(PCR); however, any other appropriate technique known in the art may beutilized. Many different methods of PCR are known in the art (e.g.,nested PCR, real time PCR). For any given screen, one of skill in theart will recognize that PCR components may be varied to achieve optimalscreening results. For example, magnesium concentration may need to beadjusted upwards when PCR is performed on disrupted alga cells to which(which chelates magnesium) is added to chelate toxic metals. Followingthe screening for clones with the proper integration of exogenousnucleic acids, clones can be screened for the presence of the encodedprotein(s) and/or products. Protein expression screening can beperformed by Western blot analysis and/or enzyme activity assays.Product screening may be performed by any method known in the art, forexample mass spectrometry, SDS PAGE protein gels, or HPLC or FPLCchromatography.

The expression of the colostrum/milk protein can be accomplished byinserting a polynucleotide sequence (gene) encoding the protein orenzyme into the chloroplast genome of a microalgae. The modified strainof microalgae can be made homoplasmic to ensure that the polynucleotidewill be stably maintained in the chloroplast genome of all descendants.A microalga is homoplasmic for a gene when the inserted gene is presentin all copies of the chloroplast genome, for example. It is apparent toone of skill in the art that a chloroplast may contain multiple copiesof its genome, and therefore, the term “homoplasmic” or “homoplasmy”refers to the state where all copies of a particular locus of interestare substantially identical. Plastid expression, in which genes areinserted by homologous recombination into all of the several thousandcopies of the circular plastid genome present in each plant cell, takesadvantage of the enormous copy number advantage over nuclear-expressedgenes to permit expression levels that can readily exceed 10% or more ofthe total soluble plant protein. The process of determining the plasmicstate of an organism of the present disclosure involves screeningtransformants for the presence of exogenous nucleic acids and theabsence of wild-type nucleic acids at a given locus of interest.

c. Vectors

Numerous suitable expression vectors are known to those of skill in theart. The following vectors are provided by way of example; for bacterialhost cells: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors,lambda-ZAP vectors (Stratagene), pTrc99a, pKK223-3, pDR540, and pRIT2T(Pharmacia); for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3,pBPV, pMSG, pET21a-d(+) vectors (Novagen), and pSVLSV40 (Pharmacia).However, any other plasmid or other vector may be used so long as it iscompatible with the host cell. For example, illustrative vectorsincluding without limitation, psbA-kanamycin vector can be used for theexpression of one or more milk/colostrum proteins, e.g., in acyanobacteria or in the plastids of a photosynthetic organism.

Knowledge of the chloroplast genome of the host organism, for example,C. reinhardtii, is useful in the construction of vectors for use in thedisclosed embodiments. Chloroplast vectors and methods for selectingregions of a chloroplast genome for use as a vector are well known (see,for example, Bock, J. Mol. Biol. 312:425-438, 2001; Staub and Maliga,Plant Cell 4:39-45, 1992; and Kavanagh et al., Genetics 152:1111-1122,1999, each of which is incorporated herein by reference). The entirechloroplast genome of C. reinhardtii is available to the public on theworld wide web, at the URL “biology.duke.edu/chlamy_genome/-chloro.html”(see “view complete genome as text file” link and “maps of thechloroplast genome” link; J. Maul, J. W. Lilly, and D. B. Stern,unpublished results; revised Jan. 28, 2002; to be published as GenBankAce. No. AF396929; and Maul, J. E., et al. (2002) The Plant Cell, Vol.14 (2659-2679)). Generally, the nucleotide sequence of the chloroplastgenomic DNA that is selected for use is not a portion of a gene,including a regulatory sequence or coding sequence. For example, theselected sequence is not a gene that if disrupted, due to the homologousrecombination event, would produce a deleterious effect with respect tothe chloroplast. For example, a deleterious effect on the replication ofthe chloroplast genome or to a plant cell containing the chloroplast. Inthis respect, the website containing the C. reinhartdii chloroplastgenome sequence also provides maps showing coding and non-coding regionsof the chloroplast genome, thus facilitating selection of a sequenceuseful for constructing a vector (also described in Maul, I. E., et al.(2002) The Plant Cell, Vol. 14 (2659-2679)). For example, thechloroplast vector, p322, is a clone extending from the Eco (Eco RI)site at about position 143.1 kb to the Xho (Xho I) site at aboutposition 148.5 kb (see, world wide web, at the URL“biology.duke.edu/chlamy_genome/chloro.html”, and clicking on “maps ofthe chloroplast genome” link, and “140-150 kb” link; also accessibledirectly on world wide web at URL “biology. duke. edu/chl am-y/chl oro/c hl oro140. html”).

For expression of the colostrum/milk polypeptide in a host, anexpression cassette or vector may be employed. The expression vectorwill comprise a transcriptional and translational initiation region,which may be inducible or constitutive, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and a transcriptional and translational terminationregion. These control regions may be native to the gene, or may bederived from an exogenous source. Expression vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences encoding exogenousproteins. A selectable marker operative in the expression host may bepresent in the vector.

The nucleotide sequences disclosed herein may be inserted into a vectorby a variety of methods. In the most common method the sequences areinserted into an appropriate restriction endonuclease site(s) usingprocedures commonly known to those skilled in the art and detailed in,for example, Green and Sambrook, Molecular Cloning, A Laboratory Manual,4th Ed., Cold Spring Harbor Press, (2012) and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons (through 2013).

Further provided are host cells that can be transformed with vectors.One of skill in the art will recognize that such transformation includestransformation with circular vectors, linearized vectors, linearizedportions of a vector, or any combination of the above. Thus, a host cellcomprising a vector may contain the entire vector in the cell (in eithercircular or linear form), or may contain a linearized portion of avector of the present disclosure.

d. Colostrum/Milk Protein Expression

To determine percent total soluble protein, immunoblot signals fromknown amounts of purified protein can be compared to that of a knownamount of total soluble protein lysate. Other techniques for measuringpercent total soluble protein are known to one of skill in the art. Forexample, an ELISA assay or protein mass spectrometry (for example, asdescribed in Varghese, R. S. and Ressom, H. W., Methods Mol. Bio. (2010)694:139-150) can also be used to determine percent total solubleprotein.

In some embodiments, the one or more colostrum/milk polypeptides areproduced in a genetically modified host cell at a level that is at leastabout 0.5%, at least about 1%, at least about 1.5%, at least about 2%,at least about 2.5%, at least about 3%, at least about 3.5%, at leastabout 4%, at least about 4.5%, or at least about 5% of the total solubleprotein produced by the cell. In other embodiments, the colostrum/milkcompound is produced in a genetically modified host cell at a level thatis at least about 0.15%, at least about 0.1%, or at least about 1% ofthe total soluble protein produced by the cell. In other embodiments,the colostrum/milk compound is produced in a genetically modified hostcell at a level that is at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,or at least about 70% of the total soluble protein produced by the cell.

Expression of the milk/colostrum polypeptides in the photosynthetic(e.g., algal) host cell can be detected using any method known in theart, e.g., including immunoassays (ELISA, Western Blot) and/or nucleicacid assays (RT-PCR). Sequences of expressed polypeptides can beconfirmed using any method known in the art (e.g., mass spectrometry).

Milk/colostrum polypeptides expressed in a photosynthetic (e.g., algal)host cell are generally properly folded without performing denaturationand refolding. Furthermore, the polypeptides expressed in thechloroplast genome are not glycosylated, so coding sequences do not needto be altered to remove glycosylation sites and glycosylated moieties donot need to be removed post-translationally.

Milk/colostrum polypeptides expressed in a photosynthetic (e.g., algalchloroplasts and cyanobacteria) host can have a phosphorylation pattern,even if different from the natively expressed protein, allows forbioactivity. Similar polypeptides expressed in the cytoplasm ofphotosynthetic organisms may not correctly phosphorylated, and thus notbiologically active. The phosphorylation machinery of chloroplasts andcyanobacteria can modified to increase or decrease the degree ofphosphorylation of a mammalian protein produced in those compartments.

e. Colostrum/Milk Protein Bioactivity

The bioactivity of the expressed colostrum milk polypeptides can bedetermined using any method known in the art. For example, lysozymebioactivity can be measured by determining the activity of cell lysatesor purified polypeptide to effect killing of gram positive bacteria(e.g, micrococcus cells). See, e.g., Ito, et al., Chem Pharm Bull(Tokyo). 1992 June; 40(6):1523-6 and Morsky, et al., Anal Biochem. 1983January; 128(1):77-85. Lactadherin bioactivity can be determined bymeasuring binding to phosphatidylserine. See, e.g., Otzen, et al.,Biochim Biophys Acta. (2012) 1818(4):1019-27; Hou, et al., Vox Sang.2011 Feb;100(2):187-95 and Dasgupta, et al., Transl Res. 2006 July;148(1):19-25. The bioactivity of osteopontin can be measured by theability of osteopontin to adhere to human embryonic 293 cells when inthe presence of the divalent cations, Mg²⁺ or Mn²⁺ but not Ca²⁺. See,e.g., Hu, et al, J Biol Chem. (1995) 270(17):9917-25; and Agnihotri, etal., J Biol Chem (2001) 276:28261-28267. CD14 bioactivity can bedetermined by measuring binding to lipopolysaccharide (LPS). See, e.g.,Wright, et al., Science. 1990 Sep. 21; 249(4975):1431-3. Cathelicidin-1activity can be determined using an antimicrobial assay and measuringluminescence. See, e.g., Sue, et al. Infect Immun. 2000 May; 68(5)2748-2755. MAA bioactivity can be determined by measuring the inductionof mucin3 expression by intestinal epithelial cells. See, e.g. Manuell.et al., Plant Biotechnology J, 2007 May; 5(3):402-12. Lingualantimicrobial peptide (LAP) and cathelicidin-1 bioactivity can bedetermined by measuring bactericidial activity. See, Tomasinsig, et al.,J Pept Sci. 2012 Feb;18(2):105-13. Alpha-lactalbumin bioactivity can bedetermined by measuring lactase synthase activity. See, Fitzgerald, etal., Anal Biochem. 1970 July; 36(1):43-61. The bioactivity of apolypeptide is determined in a test assay known in the art and thebioactivity of the test polypeptide can be compared to a positivecontrol (e.g., a known bioactive polypeptide or a native polypeptide)and a negative control (e.g., no peptide or a known biologicallyinactive polypeptide). In varying embodiments, colostrum/milkpolypeptides produced in the chloroplast of photosynthetic organisms arecharacterized by at least 50%, 75%, 85%, 90%, 95% 98%, 99% and even upto 100% of the level of bioactivity of the natural colostrum-derivedcounterpart protein.

5. Compositions

Further provided are compositions comprising the one or morecolostrum/milk polypeptides. Generally, the colostrum/milk polypeptidesneed not be purified or isolated from the host cell. A distinctadvantage of the compositions and methods described herein is thatadministration of the bioactive protein-expressing organism, withoutpurification or isolation, to a patient, e.g., a human or non-humanmammal, confers a clinical or nutritional benefit. For example,administration of photosynthetic organisms comprisingchloroplast-expressed milk/colostrum polypeptides, e.g. osteopontin, tothe gastrointestinal tract, e.g., orally, and is efficiently absorbedand assimilated into bodily tissues such as bone and immune cells.Accordingly, in varying embodiments, the compositions comprise thephotosynthetic (e.g., algal) host cells which have been engineered toexpress one or more colostrum/milk polypeptides. In varying embodiments,the compositions are edible by a mammal. The edible compositions cantake the form of a liquid or beverage (e.g., infant formula), a food, afeed (e.g., kibble), a food supplement, a nutraceutical (e.g., a pill).In varying embodiments, the compositions comprise a compressed algalcake (e.g., a compressed solid mass of algal cells), algal paste and/oralgal powder. In varying embodiments, the compositions are lyophilizedor spray dried. In some embodiments, the photosynthetic organisms (e.g.,algae) are lyophilized or spray-dried prior to the addition to an ediblecomposition, e.g., a food, beverage or tablet consumable by a mammal,e.g., a human, a canine, a feline. In some embodiments, thephotosynthetic organisms (e.g., algae) are formulated into a wet pasteprior to the addition to an edible composition, e.g., a food, beverageor tablet consumable by a mammal, e.g., a human, a canine, a feline. Insome embodiments, the photosynthetic organisms (e.g., algae) areformulated into a powder to be sprinkled onto or into an ediblecomposition, e.g., a food, beverage or tablet consumable by a mammal,e.g., a human, a canine, a feline. In some embodiments thephotosynthetic organisms (e.g., algae) are blended or mixed into anedible composition, e.g., a food, beverage or tablet consumable by amammal, e.g., a human, a canine, a feline.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Chloroplast Expression of Osteopontin

A cDNA encoding for bovine osteopontin was synthesized in C. reinhartdiichloroplast codon bias and ligated into a C. reinhartdii chloroplasttransformation vector. This vector directed the osteopontin cDNA intothe chloroplast genome via homologous recombination and allowed the cDNAto directly replace the psbA gene. This vector also contained regulatoryelements, promoters and untranslated regions (UTRs) that ensure thestable expression and translation of the osteopontin mRNA.

The transformation vector containing the osteopontin cDNA was introducedinto the chloroplast genome by first coating the vector onto 1 μM goldparticles and then shooting the gold particles into C. reinhartdii cellsthat had been plated on Tris-Acetate-phosphate (TAP) plates containing100 μg/mL kanamycin with a particle gun from Bio-Rad laboratory. Placeswere incubated in the dark for 24 hours followed by an incubation inlight with an intensity of 4000 lux for 2 weeks. Transformed algaeformed colonies following the incubation. Colonies from thetransformation was patched onto TAP plates containing 150 μg/mLkanamycin.

To ensure that colonies from algal chloroplast transformations containedour gene of interest PCR gene screens were done using a forward primer,5′-gtgctaggtaactaacgtttgattttt-3′, that anneals to the untranslatedregion of the psbA gene that is used to drive the accumulation of theosteopontin protein and a reverse primer,5′-CTGAATCACCACGACCATCATTAGC-3′, that anneals to the chloroplast codonoptimized osteopontin cDNA. The PCR yields a product that is 500 bp. Thechloroplast also contains up to 80 copies of its genome. To ensure thatthe osteopontin gene is integrated into all copies of the chloroplastgenome, a PCR screen was done to ensure that the gene that was beingreplaced was completely removed. Two sets of primers were used: 1. Acontrol set of primers to ensure that the PCR worked, amplifies the DNAthat encodes for the 16srRNA with a forward primer5′-ccgaactgaggttgggttta-3′ and a reverse primer5′-GGGGGAGCGAATAGGATTAG-3′. 2. A set of primers to amplify the MAA genethat currently resided in the psbA locus of the untransformed strainwith a forward primer 5% gtgctaggtaactaacgtttgattttt—3′ and a reverseprimer 5′-TCTTCACGTACTTGGTCACGTGTCATACC-3′. The loss of the MAA PCRproduct indicates a strain that is homoplasmic for osteopontin.

Homoplasmic cell lines were grown to a final volume of 20L and harvestedby continuous flow centrifugation. To isolate the osteopontin proteinsfrom the C. reinhartdii cell, the harvested cells were re-suspended in abuffer that contained 50 mM Tris-HCl pH8.0, 400 mM NaCl, and 0.5% Tween20. Cells were lysed at 4° C. by sonication with an amplitude of 25%with a pulse of 30 seconds followed by a rest period of 30 seconds. Thesonication cycle was repeated for a total of 16 minutes.

Once lysed, cell debris, insoluble proteins, lipids, and carbohydrateswere separated by centrifugation at 20,000 g for 15 mins at 4° C. Onceseparated soluble protein was mixed with 1 mL of anti-M2-F1 ag resin.Algal total soluble lysate and resin were allowed to mix for 1 hour.Following the binding of the osteopontin protein to the anti-M2-flagresin, resin was washed and unbound fractions removed. Osteopontinprotein was eluted from the flag resin using an elution buffer thatcontains 100 mM glycine-HCl pH3.5 and 400 mM NaCl. Elutions wereanalyzed by Western blot to ensure the presence of the protein (FIG.11). Protein was concentrated and used for Mass spectrometry LC-LC-MSanalysis to identify the protein as authentic osteopontin (FIG. 14).Mass spectrometry was used to identify if any osteopontin amino acidswere phosphorylated in the chloroplast produced protein (FIG. 14B). Anumber of amino acids could be identified as being phosphorylated, andall of these appear to be the same amino acids that are phosphorylatedin the native bovine protein (FIG. 14B). This unexpected resultdemonstrated that chloroplasts are able to recognize mammalianphosphorylation signals and correctly add phosphates at only theappropriate amino acids on the osteopontin protein. Osteopontin producedin algae by translation in the cytoplasm from a nuclear encoded gene isnot phosphorylated in algae cells.

To determine if osteopontin was bioactive a cell adhesion assay wasperformed. Increasing concentrations of osteopontin (1.7 nM tp 1700 nM)were coated in a 96-well microtiter plate. Once coated 1×10⁴ 293 kidneycells were incubated in the wells with RPMI media that was supplementedwith 10% fetal bovine serum and 2mM MgCl₂. Unbound cells were thenwashed off with PBS followed by the addition of 100 μL of RPMI mediathat was supplemented with 10% fetal bovine serum. Immediately, 10 μL ofa wst-8 reagent (Cell counting kit -8) was added to each well todetermine what percentage of cells that were bound to the plate comparedto the controls (FIG. 16). As a control a well had no cells added andanother well had the total number of cells added to represent 100%adherence.

Example 2 Chloroplast Expression of Lactadherin

A cDNA encoding for bovine lactadherin was synthesized in C. reinhartdiichloroplast codon bias and ligated into a C. reinhartdii chloroplasttransformation vector. This vector directed the lactadherin cDNA intothe chloroplast genome via homologous recombination and allowed the cDNAto directly replace the psbA gene. This vector also contained regulatoryelements, untranslated regions (UTRs) that ensure the stable expressionof the lactadherin mRNA.

The transformation vector containing the lactadherin cDNA was introducedinto the chloroplast genome by first coating the vector onto 1 μM goldparticles and then shooting the gold particles into C. reinhartdii cellsthat had been plated on Tris-Acetate-phosphate (TAP) plates containing100 μg/mL kanamycin with a particle guy from Bio-Rad laboratory. Placeswere incubated in the dark for 24 hours followed by an incubation inlight with an intensity of 4000 lux for 2 weeks. Transformed algaeformed colonies following the incubation. Colonies from thetransformation was patched onto TAP plates containing 150 μg/mLkanamycin.

To ensure that colonies from algal chloroplast transformations containedour gene of interest PCR gene screens were done using a forward primer,5′-gtgctaggtaactaacgtttgattttt-3′, that anneals to the untranslatedregion of the psbA gene that is used to drive the accumulation of thelactadherin protein and a reverse primer,5′-CCTGAAGTCCAAGCATTAACAATACC-3′, that anneals to the chloroplast codonoptimized cDNA. The PCR yields a product that is 500 bp (FIG. 12). Thechloroplast also contains up to 80 copies of its genome. To ensure thatthe gene that lactadherin is integrated into all copies of thechloroplast genome a PCR screen was done to ensure that the gene beingreplaced was completely removed. Two sets of primers were used: 1. Acontrol set of primers to ensure that the PCR worked amplifies the DNAthat encodes for the 16 srRNA with a forward primer5′-ccgaactgaggttgggttta-3′ and a reverse primer5′-GGGGGAGCGAATAGGATTAG-3′. 2. A set of primers to amplify the MAA genethat currently resided in the psbA locus of the untransformed strainwith a forward primer 5′-gtgctaggtaactaacgtttgattttt—3′ and a reverseprimer 5′-TCTTCACGTACTTGGTCACGTGTCATACC-3′. The loss of the MAA PCRproduct indicates a strain that is homoplasmic for lactadherin.

Homoplasmic cell lines were grown to a final volume of 20L and harvestedby continuous flow centrifugation. The lactadherin proteins werepurified from the C. reinhardtii cell by re-suspending the cell pelletin a buffer that contained 50 mM Tris-HCl pH8.0, 400 mM NaCl, and 0.5%Tween 20. Cells were lysed at 4° C. by sonication with an amplitude of25% with a pulse of 30 seconds followed by a rest period of 30 seconds.The sonication cycle was repeated for a total of 16 minutes.

Once lysed cell debris, insoluble proteins, lipids, and carbohydrateswere separated by centrifugation at 20,000 g for 15mins at 4° C. Onceseparated soluble protein was mixed with 1 mL of anti-M2-F1 ag resin.Algal total soluble lysate and resin were allowed to mix for 1 hour.Following the binding of the lactadherin protein to the anti-M2-flagresin, resin was washed and unbound fractions removed. Lactadherinprotein was eluted from the flag resin using an elution buffer thatcontains 100 mM glycine-HCl pH3.5 and 400 mM NaCl. Elutions wereanalyzed by Western blot to ensure the presence of the protein (FIG.12). Protein was concentrated and used for Mass spectrometry LC-LC-MSanalysis to identify the protein as lactadherin. Mass spectrometry wasalso used to identify any phosphorylated amino acids. No phosphorylatedamino acids were identified in lactadherin.

To determine whether algal chloroplast-expressed lactadherin isbioactive, an activity assay was performed. Lactadherin contains aphosphatidylserine-binding domain is required for the protein's functionin cell adhesion. FIG. 16 illustrates bioactivity of lactadherinexpressed from the chloroplast genome in binding to phosphatidylserine.Microtiter plates were coated with 3 μg/ml of phosphatidyl-L-serine inmethanol and methanol allowed to evaporate. Increasing titers oflactadherin from 30 nM up to 350 nM of lactadherin-FLAG were incubatedwith immobilized phosphatidyl-L-serine for 1 hour. Following incubationunbound protein was washed from the wells. The amount of boundFLAG-tagged protein was quantitated using anti-FLAG antibodiesconjugated to horseradish peroxidase (HRP). Lactadherin binds tophosphatidylserine, indicating that algae chloroplast expressedlactadherin is bioactive.

Example 3 Cyanobacteria Expression of Osteopontin and Mammary AssociatedSerum Amyloid A3 (MAA)

A gene coding for bovine osteopontin was placed in a DNA vector allowingfor the recombinant gene to be transcribed and subsequently translatedinto the osteopontin protein. FIG. 17 demonstrates the production ofosteopontin in the cyanobacteria Anabaena. Osteopontin protein wasdetected on the Western blots using an anti-osteopontin antibody. Lane 1contains wild-type anabaena while lanes 2 and lanes 3 contain atransgenic anabaena strain expressing a recombinant gene coding for thechloroplast codon optimized osteopontin gene. Lanes 4 and lanes 5contain a transgenic anabaena strain expressing a recombinant genecoding for the nuclear optimized osteopontin gene. Lane 6 contains atransgenic C. reinhartdii strain expressing osteopontin in thechloroplast that is serving as the positive control. FIG. 18demonstrates the expression of osteopontin in two additionalcyanobacteria strains (Leptolyngbya and Synechocystis 6803). Westernblots were probed with an anti-osteopontin antibody. Lane 1 contains atransgenic C. reinhartdii strain expressing osteopontin in thechloroplast that is serving as the positive control. Lane 2 is theprotein ladder. Lane 3 is the wild-type Leptolyngba negative control.Lane 3 and lane 4 are transgenic Leptolyngba strains expressing anuclear codon optimized osteopontin gene. Lane 5 is a wild-typeSynechocystis 6803 that is serving as a negative control. Lane 6 andlane 7 contain a transgenic Synechocystis. 6803 strain expressing arecombinant gene coding for the chloroplast codon optimized osteopontingene. Lanes 8 and lanes 9 contain a transgenic Synechocystis 6803 strainexpressing a recombinant gene coding for the nuclear optimizedosteopontin gene. FIG. 19 demonstrates the production of MAA in thecyanobacteria anabaena. Western blots were detected with an anti-MAAantibody. Lane 1 contains a transgenic C. reinhartdii strain expressingthe MAA protein in the chloroplast. Lane 2 contains wild-type anabaenawhich serves as a negative control. Lane 3 contains a transgenicanabaena strain that is expressing a nuclear codon optimized MAA gene.FIG. 20 is a Western blot demonstrating the production of MAA in thecyanobacteria, Synechococcus elongates 7942. Lane 1 contains thewild-type Synechococcus elongates 7942 serving as a negative control.Lane 2 containes a transgenic Synechococcus elongates 7942 strain thatis expressing the MAA recombinant gene that has not been induced by F41.Lane 3 containes a transgenic Synechococcus elongates 7942 strain thatis expressing the MAA recombinant gene that has been induced by F41.

Example 4 Co-Expression of Nuclear Mammary Associated Serum Amyloid A3(MAA) and Chloroplast Osteopontin in Chloroplast

A cDNA encoding for bovine osteopontin was synthesized in C. reinhartdiichloroplast codon bias and ligated into a C. reinhartdii chloroplasttransformation vector. The transformation vector containing theosteopontin cDNA was introduced into the chloroplast genome of C.reinhartdii cells by particle bombardment. Transformed algae formedcolonies following two weeks incubation.

To ensure that colonies from algal chloroplast transformations containedour gene of interest PCR gene screens were done using a forward primer,5′-gtgctaggtaactaacgtttgattitt-3′ and a reverse primer,5′-GGGGGAGCGAATAGGATTAG-3′. The PCR yields a product that isapproximately 700 bp. To ensure that the osteopontin is integrated intoall copies of the chloroplast genome a PCR screen was done to ensurethat the gene that was being replaced was completely removed. Two setsof primers were used: 1. A control set of primers to ensure that the PCRreaction worked with a forward primer 5′-ccgaactgaggttgggttta-3′ and areverse primer 5′-GGGGGAGCGAATAGGATTAG-3′ and a set of primers toamplify the MAA gene that resided in the psbA locus of the untransformedstrain with a forward primer 5′-ggaaggggaggacgtaggtacataaa-3′ and areverse primer 5′-ttagaacgtgttttgttcccaat-3′. The loss of the MAA PCRproduct indicates a strain that is homoplasmic for osteopontin.

A cDNA encoding for bovine MAA was synthesized in C. reinhartdii nuclearcodon bias and ligated into a C. reinhartdii nuclear transformationvector. The transformation vector containing the MAA cDNA was introducedinto the nuclear genome of the homoplasmic osteopontin-transformedstrain by electroporating it into C. reinhartdii cells. Transformedalgae formed colonies following the incubation. Thus, the osteopontinexpression construct was stably transformed into the chloroplast genomeand the MAA expression cassette was transformed into the nuclear genomeof the same cell.

Clones were checked by Western blot for the presence of both the MAAprotein using an anti-MAA antibody and the osteopontin protein using ananti-flag antibody (FIG. 21). Clones 1-6 of FIG. 22 demonstrate strainsof algae that produce both the MAA protein and the osteopontin protein.

The commonly owned, co-pending application International Appl. No.PCT/US2015/016596, entitled “COLOSTRUM/MILK PROTEIN COMPOSITIONS,” filedon Feb. 19, 2015 (Attorney Docket No. UCSDP033AWO/SD2014-107-3PCT) isexplicitly incorporated by reference in its entirety for its teachingsregarding expression of mammalian colostrum/milk proteins from thenucleus of a photosynthetic organism.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Informal Sequence Listing

bovine osteopontin nucleic acid sequence with optional N-terminalflag tag (underlined) for expression in the chloroplast genomeSequence ID No: 1GATTACAAAGATGATGACGATAAAAGTTTACCTGTAAAACCAACATCATCAGGTTCATCAGAAGAAAAACAATTAAATAATAAATATCCAGATGCTGTTGCAATTTGGTTAAAACCTGATCCATCACAAAAACAAACATTTTTAACACCACAAAATTCAGTATCATCAGAAGAAACAGATGATAATAAACAAAATACATTACCATCAAAATCAAATGAATCACCAGAACAAACTGATGATTTAGATGATGATGATGATAATTCACAAGATGTTAATTCAAATGATTCAGATGATGCTGAAACAACAGATGATCCTGATCATTCAGATGAATCACATCACTCAGATGAATCAGATGAAGTTGATTTTCCTACAGATATTCCAACTATTGCTGTTTTTACACCATTTATTCCTACAGAATCAGCTAATGATGGTCGTGGTGATTCAGTAGCTTATGGTTTAAAATCACGTTCAAAAAAATTTCGTCGTTCAAATGTACAATCACCAGATGCTACTGAAGAAGATTTCACATCACACATTGAATCAGAAGAAATGCACGATGCTCCAAAAAAAACTTCACAATTAACAGATCATTCAAAAGAAACTAATTCATCAGAATTATCAAAAGAATTAACACCAAAAGCTAAAGATAAAAATAAACATTCAAATTTAATTGAATCACAAGAAAATTCAAAATTATCACAAGAATTTCATTCATTAGAAGATAAATTAGATTTAGATCACAAATCAGAAGAAGATAAACATTTAAAAATTCGTATTTCACATGAATTAGATTCAGCTTCATCAGAAGTTAATbovine osteopontin amino acid sequence with optional N-terminalflag tag (underlined) Sequence ID No: 2DYKDDDDKSLPVKPTSSGSSEEKQLNNKYPDAVAIWLKPDPSQKQTFLTPQNSVSSEETDDNKQNTLPSKSNESPEQTDDLDDDDDNSQDVNSNDSDDAETTDDPDHSDESHHSDESDEVDFPTDIPTIAVFTPFIPTESANDGRGDSVAYGLKSRSKKFRRSNVQSPDATEEDFTSHIESEEMHDAPKKTSQLTDHSKETNSSELSKELTPKAKDKNKHSNLIESQENSKLSQEFHSLEDKLDLDHKSEEDKHLKIRISHELDSASSEVNbovine lactadherin nucleic acid sequence with optional N-terminalflag tag (underlined) for expression in the chloroplast genomeSequence ID No: 3GATTACAAAGATGATGACGATAAAAGTTTTTCAGGTGATTTCTGTGATTCATCACAATGTTTACATGGTGGTACATGTTTATTAAATGAAGATCGTACTCCACCATTCTATTGTTTATGTCCTGAAGGTTTTACAGGTTTATTATGTAATGAAACAGAACATGGTCCATGTTTTCCAAATCCATGTCACAATGATGCAGAATGTCAAGTTACTGATGATTCACATCGTGGTGATGTTTTTATTCAATATATTTGTAAATGTCCATTAGGTTATGTTGGTATTCACTGTGAAACAACATGTACTTCACCTTTAGGTATGCAAACTGGTGCTATTGCAGATTCACAAATTTCAGCTTCATCAATGCATTTAGGTTTTATGGGTTTACAACGTTGGGCTCCAGAATTAGCACGTTTACACCAAACAGGTATTGTTAATGCTTGGACTTCAGGTAATTATGATAAAAATCCTTGGATTCAAGTTAATTTAATGCGTAAAATGTGGGTAACAGGTGTAGTTACTCAAGGTGCTTCACGTGCAGGTTCAGCTGAATATTTAAAAACATTCAAAGTTGCATATTCAACTGATGGTCGTCAATTCCAATTCATTCAAGTTGCAGGTCGTTCAGGTGATAAAATTTTTATTGGTAATGTTAATAATTCAGGTTTAAAAATTAATTTATTCGATACTCCATTAGAAACACAATATGTTCGTTTAGTTCCTATTATTTGTCATCGTGGTTGTACTTTACGTTTTGAATTATTAGGTTGTGAATTAAATGGTTGTACAGAACCATTAGGTTTAAAAGATAATACAATTCCAAATAAACAAATTACAGCTTCATCATATTATAAAACATGGGGTTTATCAGCTTTTTCATGGTTTCCTTATTATGCTCGTTTAGATAATCAAGGTAAATTTAATGCATGGACAGCTCAAACAAATTCAGCTTCAGAATGGTTACAAATTGATTTAGGTTCACAAAAACGTGTAACAGGTATTATTACACAAGGTGCACGTGATTTTGGTCACATTCAATATGTAGCTGCATATCGTGTTGCTTATGGTGATGATGGTGTTACATGGACAGAATATAAAGATCCTGGTGCTTCAGAATCAAAAATbovine lactadherin amino acid sequence with optional N-terminalflag tag (underlined) Sequence ID No: 4DYKDDDDKSFSGDFCDSSQCLHGGTCLLNEDRTPPFYCLCPEGFTGLLCNETEHGPCFPNPCHNDAECQVTDDSHRGDVFIQYICKCPLGYVGIHCETTCTSPLGMQTGAIADSQISASSMHLGFMGLQRWAPELARLHQTGIVNAWTSGNYDKNPWIQVNLMRKMWVTGVVTQGASRAGSAEYLKTFKVAYSTDGRQFQFIQVAGRSGDKIFIGNVNNSGLKINLFDTPLETQYVRLVPIICHRGCTLRFELLGCELNGCTEPLGLKDNTIPNKQITASSYYKTWGLSAFSWFPYYARLDNQGKFNAWTAQTNSASEWLQIDLGSQKRVTGIITQGARDFGHIQYVAAYRVAYGDDGVTWTEYKDPGASESKIFPGNMDNNSHKKNIFETPFQARFVRIQPVAWHNRITLRVELLGCbovine cathelicidin-1 nucleic acid sequence with optionalN-terminal flag tag (underlined) for expression in the chloroplastgenome Sequence ID No: 5GATTACAAAGATGATGACGATAAAAGTCAAGCATTATCATATCGTGAAGCAGTTTTACGTGCTGTTGATCAATTAAATGAACAATCATCAGAACCTAATATTTATCGTTTATTAGAATTAGATCAACCTCCACAAGATGATGAAGATCCTGATTCACCTAAACGTGTATCATTTCGTGTTAAAGAAACAGTTTGTTCACGTACAACACAACAACCACCAGAACAATGTGATTTCAAAGAAAATGGTTTATTAAAACGTTGTGAAGGTACAGTAACATTAGATCAAGTACGTGGTAATTTTGATATTACTTGTAATAATCACCAATCAATTCGTATTACAAAACAACCATGGGCACCACCACAAGCAGCTCGTTTATGTCGTATTGTTGTTATTCGTGTTTGTCGTbovine cathelicidin-1 amino acid sequence with optional N-terminalflag tag (underlined) for expression in the chloroplast genomeSequence ID No: 6DYKDDDDKSQAISYREAVLRAVDQLNEQSSEPNIYRLLELDQPPQDDEDPDSPKRVSFRVKETVCSRTTQQPPEQCDFKENGLLKRCEGTVTLDQVRGNFDITCNNHQSIRITKQPWAPPQAARLCRIVVIR VCRbovine osteopontin nucleic acid sequence for expression in thechloroplast genome Sequence ID No: 7ACTTTACCTGTAAAACCAACATCATCAGGTTCATCAGAAGAAAAACAATTAAATAATAAATATCCAGATGCTGTTGCAATTTGGTTAAAACCTGATCCATCACAAAAACAAACATTTTTAACACCACAAAATTCAGTATCATCAGAAGAAACAGATGATAATAAACAAAATACATTACCATCAAAATCAAATGAATCACCAGAACAAACTGATGATTTAGATGATGATGATGATAATTCACAAGATGTTAATTCAAATGATTCAGATGATGCTGAAACAACAGATGATCCTGATCATTCAGATGAATCACATCACTCAGATGAATCAGATGAAGTTGATTTTCCTACAGATATTCCAACTATTGCTGTTTTTACACCATTTATTCCTACAGAATCAGCTAATGATGGTCGTGGTGATTCAGTAGCTTATGGTTTAAAATCACGTTCAAAAAAATTTCGTCGTTCAAATGTACAATCACCAGATGCTACTGAAGAAGATTTCACATCACACATTGAATCAGAAGAAATGCACGATGCTCCAAAAAAAACTTCACAATTAACAGATCATTCAAAAGAAACTAATTCATCAGAATTATCAAAAGAATTAACACCAAAAGCTAAAGATAAAAATAAACATTCAATTTAATTGAATCACAAGAAAAATTCAAAATTATCACAAGAATTTCATTCATTAGAAGATAAATTAGATTTAGATCACAAATCAGAAGAAGATAAACATTTAAAAATTCGTATTTCACATGAATTAGATTCAGCTTCATCAGAAGTTAATbovine osteopontin amino acid sequence The N-terminal methionine(M) and signal peptide are optionally absent. Ref. Seq. No. NP_776612.1.Sequence ID No: 8MLPVKPTSSGSSEEKQLNNKYPDAVAIWLKPDPSQKQTFLTPQNSVSSEETDDNKQNTLPSKSNESPEQTDDLDDDDDNSQDVNSNDSDDAETTDDPDHSDESHMSDESDEVDFPTDIPTIAVFTPFIPTESANDGRGDSVAYGLKSRSKKFRRSNVQSPDATEEDFTSHIESEEMHDAPKKTSQLTDHSKETNSSELSKELTPKAKDKNKHSNLIESQENSKLSQEFHSLEDKLDLDHKSEEDKHLKIRISHELDSASSEVNbovine lactadherin nucleic acid sequence for expression in thechloroplast genome Sequence ID No: 9AGTTTTTCAGGTGATTTCTGTGATTCATCACAATGTTTACATGGTGGTACATGTTTATTAAATGAAGATCGTACTCCACCATTCTATTGTTTATGTCCTGAAGGTTTTACAGGTTTATTATGTAATGAAACAGAACATGGTCCATGTTTTCCAAATCCATGTCACAATGATGCAGAATGTCAAGTTACTGATGATTCACATCGTGGTGATGTTTTTATTCAATATATTTGTAAATGTCCATTAGGTTATGTTGGTATTCACTGTGAAACAACATGTACTTCACCTTTAGGTATGCAAACTGGTGCTATTGCAGATTCACAAATTTCAGCTTCATCAATGCATTTAGGTTTTATGGGTTTACAACGTTGGGCTCCAGAATTAGCACGTTTACACCAAACAGGTATTGTTAATGCTTGGACTTCAGGTAATTATGATAAAAATCCTTGGATTCAAGTTAATTTAATGCGTAAAATGTGGGTAACAGGTGTAGTTACTCAAGGTGCTTCACGTGCAGGTTCAGCTGAATATTTAAAAACATTCAAAGTTGCATATTCAACTGATGGTCGTCAATTCCAATTCATTCAAGTTGCAGGTCGTTCAGGTGATAAAATTTTTATTGGTAATGTTAATAATTCAGGTTTAAAAATTAATTTATTCGATACTCCATTAGAAACACAATATGTTCGTTTAGTTCCTATTATTTGTCATCGTGGTTGTACTTTACGTTTTGAATTATTAGGTTGTGAATTAAATGGTTGTACAGAACCATTAGGTTTAAAAGATAATACAATTCCAAATAAACAAATTACAGCTTCATCATATTATAAAACATGGGGTTTATCAGCTTTTTCATGGTTTCCTTATTATGCTCGTTTAGATAATCAAGGTAAATTTAATGCATGGACAGCTCAAACAAATTCAGCTTCAGAATGGTTACAAATTGATTTAGGTTCACAAAAACGTGTAACAGGTATTATTACACAAGGTGCACGTGATTTTGGTCACATTCAATATGTAGCTGCATATCGTGTTGCTTATGGTGATGATGGTGTTACATGGACAGAATATAAAGATCCTGGTGCTTCAGAATCAAAAATbovine lactadherin amino acid sequence. Ref Seq No. NP 788783.1Sequence ID No: 10FSGDFCDSSQCLHGGTCLLNEDRTPPFYCLCPEGFTGLLCNETEHGPCFPNPCHNDAECQVTDDSHRGDVFIQYICKCPLGYVGIHCETTCTSPLGMQTGAIADSQISASSMHLGFMGLQRWAPELARLHQTGIVNAWTSGNYDKNPWIQVNLMRKMWVTGVVTQGASRAGSAEYLKTFKVAYSTDGRQFQFIQVAGRSGDKIFIGNVNNSGLKINLFDTPLETQYVRLVPIICHRGCTLRFELLGCELNGCTEPLGLKDNTIPNKQITASSYYKTWGLSAFSWFPYYARLDNQGKFNAWTAQTNSASEWLQIDLGSQKRVTGIITQGARDFGHIQYVAAYRVAYGDDGVTWTEYKDPGASESKIFPGNMDNNSHKKNIFETPFQARFVRIQPVAWHNRITLRVELLGCbovine cathelicidin-1 nucleic acid sequence for expression inthe chloroplast genome Sequence ID No: 11AGTCAAGCATTATCATATCGTGAAGCAGTTTTACGTGCTGTTGATCAATTAAATGAACAATCATCAGAACCTAATATTTATCGTTTATTAGAATTAGATCAACCTCCACAAGATGATGAAGATCCTGATTCACCTAAACGTGTATCATTTCGTGTTAAAGAAACAGTTTGTTCACGTACAACACAACAACCACCAGAACAATGTGATTTCAAAGAAAATGGTTTATTAAAACGTTGTGAAGGTACAGTAACATTAGATCAAGTACGTGGTAATTTTGATATTACTTGTAATAATCACCAATCAATTCGTATTACAAAACAACCATGGGCACCACCACAAGCAGCTCGTTTATGTCGTATTGTTGTTATTCGTGTTTGTCGTbovine cathelicidin-1 amino acid sequence. Ref. Seq. No. NP_777250.1Sequence ID No: 12QALSYREAVLRAVDQLNEQSSEPNIYRLLELDQPPQDDEDPDSPKRVSFRVKETVCSRTTQQPPEQCDFKENGLLKRCEGTVTLDQVRGNFDITCNNHQSIRITKQPWAPPQAARLCRIVVIRVCR*bovine milk lysozyme amino acid sequence Sequence ID No: 13KKFQRCELARTLKKLGLDGYRGVSLANWVCLARWESNYNTRATNYNRGDKSTDYGIFQINSRWWCNDGKTPKAVNACRIPCSALLKDDITQAVACAKRVVRDPQGIKAWVAWRNKCQNRDLRSYVQGCRVbovine alpha-lactalbumin amino acid sequence Sequence ID No: 14EQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIMCVKKILDKVGINYWLAHKALCSEKLDQWLCEKLbovine lingual antimicrobial peptide amino acid sequenceSequence ID No: 15 VRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRKbovine soluble CD14 amino acid sequence Sequence ID No: 16DTTEPCELDDDDFRCVCNFTDPKPDWSSAVQCMVAVEVEISAGGRSLEQFLKGADTNPKQYADTIKALRVRRLKLGAAQVPAQLLVAVLRALGYSRLKELTLEDLEVTGPTPPTPLEAAGPALTTLSLRNVSWTTGGAWLGELQQWLKPGLRVLNIAQAHSLAFPCAGLSTFEALTTLDLSDNPSLGDSGLMAALCPNKFPALQYLALRNAGMETPSGVCAALAAARVQPQSLDLSHNSLRVTAPGATRCVWPSALRSLNLSFAGLEQVPKGLPPKLSVLDLSCNKLSREPRRDELPEVNDLTLDGNPFLDPGALQHQNDPMISGVVPACARSALTMGVSGALALLQGARGFAhuman osteopontin nucleic acid sequence for expression in thechloroplast genome Sequence ID No: 17GCT ATT CCA GTT AAA CAA GCA GAC TCT GGT TCA AGT GAA GAA AAA CAATTA TAT AAT AAA TAC CCA GAT GCT GTT GCT ACA TGG TTA AAT CCT GATCCT TCA CAA AAA CAA AAT TTA TTA GCT CCA CAA ACT TTA CCT TCA AAATCT AAT GAA AGT CAT GAT CAC ATG GAT GAC ATG GAC GAC GAA GAT GACGAT GAC CAT GTA GAC TCT CAA GAT AGT ATT GAC TCA AAT GAT TCA GATGAC GTA GAT GAC ACT GAC GAC TCA CAT CAA TCA GAC GAA TCT CAT CATAGT GAT GAA TCT GAC GAA CTT GTA ACA GAT TTC CCA ACT GAT TTA CCAGCT ACT GAA GTT TTC ACA CCA GTA GTT CCA ACT GTT GAT ACT TAC GACGGT CGT GGT GAT TCT GTA GTT TAT GGT TTA CGT TCT AAA TCA AAA AAATTT CGT CGT CCT GAT ATT CAA TAT CCA GAC GCA ACT GAC GAA GAT ATTACA TCA CAC ATG GAA TCT GAA GAA TTA AAT GGT GCT TAC AAA GCT ATTCCT GTA GCA CAA GAT TTA AAT GCT CCT TCA GAC TGG GAT TCT CGT GGTAAA GAC AGT TAC GAA ACT TCA CAA CTT GAT GAT CAA AGT GCT GAA ACACAT TCA CAC AAA CAA TCT CGT CTT TAT AAA CGT AAA GCT AAT GAT GAAAGT AAT GAA CAC TCA GAT GTT ATT GAC TCA CAA GAA CTT TCT AAA GTATCA CGT GAA TTT CAC AGT CAC GAA TTT CAT TCT CAC GAA GAT ATG TTAGTT GTT GAT CCA AAA AGT AAA GAA GAA GAC AAA CAC CTT AAA TTT CGTATT TCT CAC GAA TTA GAC TCA GCA TCA TCT GAA GTT AAT TAAhuman osteopontin amino acid sequence (NCBI Reference Sequence:NP_000573.1). The N-terminal methionine (M) and signal peptideare optionally absent. Sequence ID No: 18MRIAVICFCL LGITCAIPVK QADSGSSEEK QLYNKYPDAV ATWLNPDPSQ KQNLLAPQTLPSKSNESHDH MDDMDDEDDD DHVDSQDSID SNDSDDVDDT DDSHQSDESH HSDESDELVTDFPTDLPATE VFTPVVPTVD TYDGRGDSVV YGLRSKSKKF RRPDIQYPDA TDEDITSHMESEELNGAYKA IPVAQDLNAP SDWDSRGKDS YETSQLDDQS AETHSHKQSR LYKRKANDESNEHSDVIDSQ ELSKVSREFH SHEFHSHEDM LVVDPKSKEE DKHLKFRISH ELDSASSEVNcanine osteopontin nucleic acid sequence for expression in thechloroplast genome Sequence ID No: 19GGT ATT CCA ATT AAA CAC GCA GAT AGT GGT TCA TCA GAA GAA AAA CAATTA TAC AAC AAA TAC CCT GGT GCT GTT GCT ACA TGG TTA AAA CCA GATCCT TCA CAA AAA CAA ACA TTT TTA GCT TTA CAA AAT GCT GTT TTA ACAGAA GAA ACT GAC GAC TTC AAA CAA AAA ACA TTT TCT TCA AAA TCT AACGAA AGT CAT GAC GAC GTT GAT GAA GAT GAT GGT GAT GAC GTT GAT AGTCAA GAT TCA GTT GAT TCA AAC GAC TTA GAC GAT GAT TCA AAT GAA TCAGAT GAA AGT GAT GAA TTA GTA ACA GAT TTC CCA ACT GAT ATT CCT GCAACT CAA TTA TTC ACA CCA GCT GTT CCA ACA CGT GGT AGT TAC GAC GGTCGT GGT GAT TCT GTA GCT TAT GGT TTA CGT TCA AAA TCA AAA AAA TCACAC AAA TAT GAA GTT CAA TAC CCA GAC TCA ACT GAA GAA GAT TTT ACATCA TTA GTA AAA TCT GCA TCA ATG GAA GAC GAC TTT AAT GCA GTA TTATTA AGT CGT ACT GTT CGT GGT ACT TCA GAT CGT GAT TCA CAC GCT AAAGAC TCT CAA GAA ACT TCA CAA TTA GAT GAT CAT TCT ATG GAA ACT AAAGGT CGT AAA CAC TCA CAA GAA TAC AAA TTA CGT GCT AGT GAC GAA TCAAAT ATG CAC AGT CAC GAA ATT GGT TCA CAA GAA AAT TCT GAA GTA TCTAGT GAA TTA GTT AGT CAA TTA AGT CAA TCA CAC GAA AAA GAA TTA ATTGTT GAC TCT AAA AGT GAA GAA GAA GAT AAA CAC TTA AAA TTT CAT GTTTCT CAC GAA TTA GAT AGT GCT TCA AGT GAA ATT ATT taa tct agacanine osteopontin amino acid sequence (NCBI Reference Sequence:XP_003434072.1). The N-terminal methionine (M) and signalpeptide are optionally absent. Sequence ID No: 20MRIAVICFCL LGIAYAIPIK HADSGSSEEK QLYNKYPGAV ATWLKPDPSQ KQTFLALQNAVLTEETDDFK QKTFSSKSNE SHDDVDEDDG DDVDSQDSVD SNDLDDDSNE SDESDELVTDFPTDIPATQL FTPAVPTRGS YDGRGDSVAY GLRSKSKKSH KYEVQYPDST EEDFTSLVKSASMEDDFNAV LLSRTVRGTS DRDSHAKDSQ ETSQLDDHSM ETKGRKHSQE YKLRASDESNMHSHEIGSQE NSEVSSELVS QLSQSHEKEL IVDSKSEEED KHLKFHVSHE LDSASSEINfeline osteopontin nucleic acid sequence for expression in thechloroplast genome Sequence ID No: 21GGT ATT CCA ATT AAA CAA ACA GAC AGT GGA TCT AGT GAA GAA AAA CAATTA TAT AAC AAA TAT CCT GTT GCT GTA GCT ACT TGG CCA AAA CCA GATCCT TCT CAA AAA CAA ACT TTT TTA GCT TTA CAA AAC GCA GTT TTA TCTGAA GAA ACA GAT GAT TTC AAA CAA AAA ACA TTA GCA TCA AAA TCT AACGAA TCA CAT GAT GTA GAT GAC GAA GAC GAT GAA GAC GAC GTA GAT TCTCAA GAT TCT GTT GAT TCT CAC GAT ACA GAT GAT GAT AGT AAT CCA AGTGAC GAA AGT GAT GAA CTT GTA ACA GAC TTT CCA ACT GAC GTA CCA GCTACT CAA TTC TTT ACA CCA GCT GTT CCA ACT CGT GAT AGT TAT GAC GGACGT GGT GAC TCT GTT GCA TAC GGT CTT CGT TCA AAA TCA AAA AAA TCACAT CGT TAC GAA GAT CAA TAT CCA GAT TCA ACA GAA GAA GAC TTT ACATCT TTA GTA AAA AGT CAA TCA ATG GAA GAT GAT TTT AAT GCT GTA CTTTTA AGT CAT ACA GTT CGT CGT TCT CCT GAC CGT GAT TCA CAT GTT AAAGAT TCA CAA GAA ACT TCA CAA GTT GAT GAC CAC TCT ATG GAA ACA AAAAGT CGT AAA CAC TCT AAA GAA TAC AAA TTA AAA GCT TCT GAT GAA AATAAT AAA CAC AGT CAC GAA ATT GGT TCT CAA GAA TCT TCT GAC ATT TCTAGT GAA TTA GTA GGT CAA ACT GTT CAA TCT AAT GAA AAA GAA CTT GTTCAA CAC CCA GAA AGT GAA GAA CAA GAT AAA CAC TTA AAA TTT CGT GTTTCA CAT GAA TTA GAT TCA GCA TCA AGT GAA GTT AAT TAAfeline osteopontin amino acid sequence (NCBI Reference Sequence:XP_003985233.1). The N-terminal methionine (M) and signalpeptide are optionally absent. Sequence ID No: 22MRIAVICFCL LGIAYAIPIK QTDSGSSEEK QLYNKYPVAV ATWPKPDPSQ KQTFLALQNAVLSEETDDFK QKTLASKSNE SHDVDDEDDE DDVDSQDSVD SHDTDDDSNQ SDESDELVTDFPTDVPATQF FTPAVPTRDS YDGRGDSVAY GLRSKSKKSH RYEDQYPDST EEDFTSLVKSQSMEDDFNAV LLSHTVRRSP DRDSHVKDSQ ETSQVDDHSM ETKSRKHSKE YKLKASDENNKHSHEIGSQE SSDISSELVG QTVQSNEKEL VQHPESEEQD KHLKFRVSHE LDSASSEVNplastid retention nucleic acid sequence Sequence ID No: 23atggccgtcatgatgcgcacccaggcgcccgctgccactcgcgcttcatcgcgcgtcgctgttgccgctcgcccggctgctcgccgcgccgtggtggtccgcgccgaggctplastid retention amino acid sequence Sequence ID No: 24MAVMMRTQAPAATRASSRVAVAARPAARRAVVVRAEA

1-73. (canceled)
 74. A microalgal chloroplast comprising apolynucleotide encoding mammalian lactadherin integrated into thechloroplast genome, wherein the lactadherin polypeptide comprises atleast about 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:10,wherein the chloroplast expresses a bioactive lactadherin polypeptidethat is not glycosylated.
 75. The chloroplast of claim 74, wherein thechloroplast is from a green algae.
 76. The chloroplast of claim 75,wherein the green algae is selected from the group consisting ofChlamydomonas, Dunaliella, Haematococcus, Chlorella, and Scenedesmaceae.77. The chloroplast of claim 74, wherein the microalgal chloroplast isfrom a microalga selected from the group consisting of Chlamydomonasreinhartdii, Dunaliella salina, Haematococcus pluvialis, Chlorellavulgaris, Acutodesmus obliquus, and Scenedesmus dimorphus.
 78. Thechloroplast of claim 74, wherein the lactadherin polypeptide is from amammal selected from the group consisting of human, canine, feline,bovine, porcine, ovine, and caprine.
 79. The chloroplast of claim 74,wherein the polynucleotide encoding mammalian lactadherin comprises apolynucleotide having at least about 85%, 90%, 95%, 98% or 99% sequenceidentity sequence identity to SEQ ID NO:9.
 80. A cell comprising thechloroplast of claim
 74. 81. The cell of claim 80, wherein the cell isintact.
 82. The cell of claim 80, wherein the cell is dried.
 83. Thecell of claim 82, wherein the cell is freeze-dried.
 84. The cell ofclaim 80, wherein the cell is a microalgal cell.
 85. The cell of claim80, wherein the cell is from a green algae selected from the groupconsisting of Chlamydomonas, Dunaliella, Haematococcus, Chlorella, andScenedesmaceae.
 86. A method for producing one or more mammaliancolostrum or milk proteins, comprising culturing the cell of claim 80.87. A composition edible by a mammal and/or a chicken comprising one ormore populations of cells of claim
 80. 88. The composition of claim 87,wherein the composition is selected from a beverage, a food, a feed, afood supplement, and a nutraceutical.
 89. The composition of claim 87,wherein the composition is selected from the group consisting of acompressed algal cake, an algal paste, and an algal powder.
 90. Thecomposition of claim 87, wherein the composition is dried.
 91. Thecomposition of claim 90, wherein the composition is freeze-dried,lyophilized, or spray-dried.